Hepatitis b virus typing and resistance assay

ABSTRACT

The present invention provides methods, kits, and oligonucleotides for detecting and analyzing the nucleotide sequence of a reverse transcriptase (RT) region of the polymerase (Pol) gene of Hepatitis B Virus (HBV). In certain embodiments, a target RT region is amplified and subjected to DNA sequencing. The sequence obtained is compared to one or more DNA sequences characteristic of an HBV genotype or serotype, and/or one or more DNA sequences characteristic of an HBV mutation that confers resistance to a drug or vaccine, to determine the HBV genotype or serotype of the amplified product and/or the presence or absence of one or more DNA sequences characteristic of an HBV mutation that confers resistance to a drug or vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/774,960, filed Feb. 22, 2013, which claims the benefit of U.S.provisional application No. 61/605,029, filed Feb. 29, 2012, which ishereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates generally to the area of Hepatitis B Virus(HBV) detection and characterization. In particular, the inventionrelates to methods and compositions for determining HBVgenotype/serotype and for detecting mutations associated with drugresistance and/or vaccine escape.

BACKGROUND OF THE INVENTION

More than 400 million people are chronically infected with HBV, a small,circular, partially double-stranded DNA virus of approximately 3200 basepairs. Chronic infection can result in cirrhosis (scarring) of theliver, liver cancer, liver failure, and death. Current treatment optionsfor chronically infected HBV patients include interferon, peginterferon,and antiviral drugs, such as lamivudine, adefovir, entecavir,telbivudine, and tenofovir targeted against the polymerase (Pol) regionof HBV.

The genome of HBV is made of circular DNA, but it is unusual because theDNA is not fully double-stranded. One end of the full-length strand islinked to the viral DNA polymerase. The genome is 3020-3320 nucleotideslong (for the full-length strand) and 1700-2800 nucleotides long (forthe short length-strand). The negative-sense, (non-coding), iscomplementary to the viral mRNA. The viral DNA is found in the nucleussoon after infection of the cell. The partially double-stranded DNA isrendered fully double-stranded by completion of the (+) sense strand andremoval of a protein molecule from the (−) sense strand and a shortsequence of RNA from the (+) sense strand. Non-coding bases are removedfrom the ends of the (−) sense strand and the ends are rejoined. Thereare four known genes encoded by the genome, called C, X, P, and S. Thecore protein is coded for by gene C (HBcAg), and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBeAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P (Pol). Gene Sis the gene that codes for the surface antigen (HBsAg). The HBsAg geneis one long open reading frame but contains three in frame start (ATG)codons that divide the gene into three sections, pre-S1, pre-S2, and S.Because of the multiple start codons, polypeptides of three differentsizes called large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S)are produced. Because of the small size of the genome, many of the openreading frames overlap. For example the S open reading frame overlapswith the Pol open reading frame, specifically, HBV nucleotide positions155 to 832, coding 226 amino acids of the surface antigen, overlaps withthe RT region, nucleotide positions 130 to 1161, of the Pol gene. Drugresistance is associate with mutations in the RT region.

The virus is divided into four major serotypes (adr, adw, ayr, ayw)based on antigenic epitopes presented on its envelope proteins, and intonine genotypes (A-I) according to overall nucleotide sequence variationof the genome. The genotypes have a distinct geographical distributionand are used in tracing the evolution and transmission of the virus.Differences between genotypes affect the disease severity, course andlikelihood of complications, and response to treatment and vaccination.Genotypes differ by at least 8% of their sequence and were firstreported in 1988 when six were initially described (A-F). Norder H,Courouce A M, Magnius L O (1994). “Complete genomes, phylogenicrelatedness and structural proteins of six strains of the hepatitis Bvirus, four of which represent two new genotypes.” Virology 198 (2):489-503 (incorporated herein by reference for the description of HBVgenotypes). Three further types have since been described (G, H, and I).Shibayama T, Masuda G, Ajisawa A, Hiruma K, Tsuda F, Nishizawa T,Takahashi M, Okamoto H (May 2005). “Characterization of seven genotypes(A to E, G and H) of hepatitis B virus recovered from Japanese patientsinfected with human immunodeficiency virus type 1.” Journal of MedicalVirology 76 (1): 24-32 (incorporated herein by reference for thedescription of HBV genotypes). “A complex hepatitis B virus (X/C)recombinant is common in Long An county, Guangxi and may have originatedin southern China.” Journal of General Virology (2011),92,402-411Journal of General Virology (2011), 92, 402-411. Mostgenotypes are now divided into subgenotypes with distinct properties.Schaefer S (January 2007). “Hepatitis B virus taxonomy and hepatitis Bvirus genotypes.” World Journal of Gastroenterology: WJG 13 (1): 14-21(incorporated herein by reference for the description of HBV genotypes).

HBV has a high degree of genetic variation with nine known genotypes(A-I). Different mutations in the various HBV genotypes are associatedwith how the virus can escape the immune system or become resistant toantiviral drugs. Ray, K, “Hepatitis: Genetic variability in HBVresistance.” Nature Reviews Gastroenterology and Hepatology 8, 535(October 2011) (incorporated herein by reference for the description ofHBV mutations associated with immune system/vaccine escape and drugresistance).

SUMMARY OF THE INVENTION

In certain embodiments, the invention provides a method for detectingand analyzing the nucleotide sequence of a reverse transcriptase (RT)region of the polymerase (Pol) gene of Hepatitis B Virus (HBV). Themethod entails:

-   -   contacting a nucleic acid sample with a primer pair specific for        a target RT region and carrying out a real-time amplification        reaction to produce and quantify an amplified product if the        target RT region is present in the sample;    -   determining the DNA sequence of the amplified product; and    -   comparing the DNA sequence of the amplified product to:        -   one or more DNA sequences characteristic of an HBV genotype            or serotype; and/or        -   one or more DNA sequences characteristic of an HBV mutation            that confers resistance to a drug or vaccine;    -   to determine the HBV genotype or serotype of the amplified        product and/or the presence or absence of one or more DNA        sequences characteristic of an HBV mutation that confers        resistance to a drug or vaccine. In particular embodiments, the        amplified product includes a nucleotide sequence that encodes an        amino acid sequence including RT53 through RT256, as numbered        from the N-terminus of the RT domain.

In certain embodiments, the DNA sequence of the amplified product isdetermined using:

-   -   a first forward primer that anneals to the HBV genome 5′ of        nucleotide 286;    -   a first reverse primer that anneals to the HBV genome 3′ of        nucleotide 895;    -   a second forward primer that anneals to the HBV genome between        nucleotide 377 and nucleotide 827; and    -   a second reverse primer that anneals to the HBV genome between        nucleotide 377 and nucleotide 827. In various embodiments, the        primer pair specific for a target RT region includes: the first        forward primer and the first reverse primer; the first forward        primer and the second reverse primer; the second forward primer        and the first reverse primer; or the second forward primer and        the second reverse primer.

The method can additionally include determining amount of amplifiedproduct produced and diluting the amplified product by 1:2 to 1:8 priorto DNA sequencing. In various embodiments, the degree of dilution is1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or falls within a range bounded by anyof these values. In certain embodiments, the method included dilutingthe amplified product by either 1:2 or 1:5 prior to sequencing. Inparticular embodiments, the amplification reaction is a real-timeamplification method that is employed to determine the amount ofamplified product produced.

In certain embodiments, the amplified product includes at least 270nucleotides. In particular embodiments, the amplified product includesfewer than 1200 nucleotides, e.g., the amplified product can includebetween 1000 and 1050 nucleotides.

In various embodiments, the method employs one or more of: a primer thatanneals to the HBV genome between nucleotide 180 and nucleotide 204; aprimer that anneals to the HBV genome between nucleotide 1178 andnucleotide 1202; a primer that anneals to the HBV genome betweennucleotide 420 and nucleotide 450; and/or a primer that anneals to theHBV genome between nucleotide 673 and nucleotide 704. Any of theabove-described primers can be at least 24 nucleotides in length. Invarious embodiments, the method employs one or more of: a primer thathas a nucleotide sequence including SEQ ID NO:1; a primer that has anucleotide sequence including SEQ ID NO:2; a primer that has anucleotide sequence including SEQ ID NO:3; a primer that has anucleotide sequence including SEQ ID NO:4.

In illustrative embodiments, at least two primers are employed, and thetwo primers have nucleotide sequences including: SEQ ID NO:1 and SEQ IDNO:2; SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:1 and SEQ ID NO:4; SEQ IDNO:2 and SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:4; or SEQ ID NO:3 andSEQ ID NO:4.

In further illustrative embodiments, at least three primers areemployed, and the three primers have nucleotide sequences including: SEQID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:2, SEQ ID NO:3, and SEQID NO:4; SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:4; or SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQID NO:4.

In a specific embodiment, at least four primers are employed, and thefour primers have nucleotide sequences including SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4.

In certain embodiments of the method, a probe is employed to determinethe amount of amplified product produced for a sequencing assay. Theprobe can be, e.g., at least 22 nucleotides in length. In anillustrative embodiment, the probe has a nucleotide sequence includingSEQ ID NO:5. In particular embodiments, the probe includes a fluorophoreand a quencher at either end of the molecule.

In certain embodiments, the amplification reaction includes polymerasechain reaction (PCR), e.g., real-time polymerase chain reaction(RT-PCR). In particular embodiments, the DNA sequence is determined bycycle DNA sequencing.

In certain embodiments, the nucleic acid sample is obtained from a humanpatient. In this case, the method can additionally entail recording theHBV genotype or serotype and/or any HBV mutation and/or HBV vaccineescape in a patient medical record, e.g., recording the HBV genotype orserotype and/or any HBV mutation and/or HBV vaccine escape in acomputer-readable medium. In various embodiments, the patient medicalrecord is maintained by a laboratory, physician's office, a hospital, ahealth maintenance organization, an insurance company, or a personalmedical record website. In various embodiments, the one or more DNAsequences characteristic of an HBV mutation that confers resistance to adrug include sequences characteristic of HBV mutations that conferresistance to lamivudine, adefovir, entecavir, telbivudine, tenofovir,or any combination thereof. In particular embodiments, the method canadditionally include prescribing, initiating, and/or altering therapyfor HBV or initiating and/or altering an HBV vaccine therapy. Forexample, when an HBV mutation that confers resistance to a drug is foundto be present in a sample from a patient, the method can includeprescribing and/or administering a different drug to the patient.Likewise, when an HBV mutation associated with vaccine escape is foundto be present in a sample from a patient, the method can includedetermining that the patient is not a candidate for treatment with thatvaccine.

In particular embodiments, the method has one or more of the followingperformance characteristics:

The method produces a sequencing result in 95% or more of specimenscontaining HBV DNA at a concentration of at least 200-400 IU/mL, e.g.,at least 200 or at least 400 IU/mL, HBV DNA.

The method has an analytical specificity of 99.5% or greater, e.g.,100.0%, calculated using the frequency of repeatedly reactive results.

The method is capable of detecting mixed bases more than 50% of thetime, when the two populations of bases are at equal concentration, forthe mixture panel at the 1×10⁵ IU/mL viral load level.

Another aspect of the invention is a kit for detecting and analyzing thenucleotide sequence of a reverse transcriptase (RT) region of thepolymerase (Pol) gene of Hepatitis B Virus (HBV). In certainembodiments, the kit includes:

-   -   a primer that anneals to the HBV genome 5′ of nucleotide 286;    -   a primer that anneals to the HBV genome 3′ of nucleotide 895;        and    -   two primers that anneal to the HBV genome between nucleotide 377        and nucleotide 827.

In particular embodiments, each of the primers is provided in a separatecontainer, and the kit further includes an additional containerincluding a primer pair specific for a target RT region.

In certain embodiments, two of the primers define a target RT regionthat includes at least 270 nucleotides. In particular embodiments, thetarget RT region includes fewer than 1200 nucleotides, e.g., the targetRT region can include between 1000 and 1050 nucleotides.

In various embodiments, the kit includes one or more of: a primer thatanneals to the HBV genome between nucleotide 180 and nucleotide 204; aprimer that anneals to the HBV genome between nucleotide 1178 andnucleotide 1202; a primer that anneals to the HBV genome betweennucleotide 420 and nucleotide 450; and/or a primer that anneals to theHBV genome between nucleotide 673 and nucleotide 704. Any of theabove-described primers can be at least 24 nucleotides in length. Invarious embodiments, the kit includes one or more of: a primer that hasa nucleotide sequence including SEQ ID NO:1; a primer that has anucleotide sequence including SEQ ID NO:2; a primer that has anucleotide sequence including SEQ ID NO:3; a primer that has anucleotide sequence including SEQ ID NO:4.

In illustrative embodiments, the kit includes at least two primers, andthe two primers have nucleotide sequences including: SEQ ID NO:1 and SEQID NO:2; SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:1 and SEQ ID NO:4; SEQID NO:2 and SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:4; or SEQ ID NO:3 andSEQ ID NO:4.

In further illustrative embodiments, the kit includes at least threeprimers, and the three primers have nucleotide sequences including: SEQID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:2, SEQ ID NO:3, and SEQID NO:4; SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:4; or SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQID NO:4.

In a specific embodiment, the kit includes at least four primers, andthe four primers have nucleotide sequences including SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4.

In certain embodiments of the method, the kit includes a probe thatanneals to the target RT region. For example, the probe can be presentin the additional container including the primer pair specific for atarget RT region. The probe can be, e.g., at least 22 nucleotides inlength. In an illustrative embodiment, the probe has a nucleotidesequence including SEQ ID NO:5. In particular embodiments, the probeincludes a fluorophore and a quencher at either end of the molecule.

Another aspect of the invention is oligonucleotides useful, e.g., asprimers and/or probes in the methods described herein. In variousembodiments, an oligonucleotide of the invention has a nucleotidesequence consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ IDNO:4; or SEQ ID NO:5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HBV genome and positions of primers. “FP”=forward primer;“RP”=reverse primer; “nt” followed by a number refers to the nucleotidenumber, numbered from the 5′ end of the full-length strand of the HBVgenome; “RT” followed by a number refers to the amino acid number,numbered from the N-terminus of the RT domain in the HBV polymeraseprotein; “s” followed by a number refers to the amino acid number,numbered from the N-terminus of the HBV surface antigen protein (i.e.,the “small” or “S” protein).

FIG. 2: Schematic of Example 1's HBV Sequencing Assay workflow.

DETAILED DESCRIPTION

In certain embodiments, the present invention provides oligonucleotideprimers and a fluorescently-labeled probe, as well as a novel method fordetermining the DNA sequence of the reverse transcriptase (RT) region ofthe polymerase (Pol) gene of Hepatitis B Virus (HBV). In this assay, thenucleotide sequence of the HBV isolate is analyzed for two purposes: todetermine the genotype of HBV and to determine if mutations associatedwith drug resistance are present in the isolate. Both types ofinformation are useful in the management of patients with chronic HBV.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “nucleic acid” refers to a nucleotide polymer, and unlessotherwise limited, includes known analogs of natural nucleotides thatcan function in a similar manner (e.g., hybridize) to naturallyoccurring nucleotides.

The term nucleic acid includes any form of DNA or RNA, including, forexample, genomic DNA; complementary DNA (cDNA), which is a DNArepresentation of mRNA, usually obtained by reverse transcription ofmessenger RNA (mRNA) or by amplification; DNA molecules producedsynthetically or by amplification; and mRNA.

The term nucleic acid encompasses double- or triple-stranded nucleicacids, as well as single-stranded molecules. In double- ortriple-stranded nucleic acids, the nucleic acid strands need not becoextensive (i.e, a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modificationthereof, such as by methylation and/or by capping. Nucleic acidmodifications can include addition of chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the individual nucleic acid bases orto the nucleic acid as a whole. Such modifications may include basemodifications such as 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbonemodifications, unusual base pairing combinations such as the isobasesisocytidine and isoguanidine, and the like.

More particularly, in certain embodiments, nucleic acids, can includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing nonnucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleic acid also encompasses linkednucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499,6,670,461, 6,262,490, and 6,770,748, which are incorporated herein byreference in their entirety for their disclosure of LNAs.

The nucleic acid(s) can be derived from a completely chemical synthesisprocess, such as a solid phase-mediated chemical synthesis, from abiological source, such as through isolation from any species thatproduces nucleic acid, or from processes that involve the manipulationof nucleic acids by molecular biology tools, such as DNA replication,PCR amplification, reverse transcription, or from a combination of thoseprocesses.

The term “target nucleic acids” is used herein to refer to particularnucleic acids to be detected in the methods of the invention.

As used herein the term “target nucleotide sequence” refers to amolecule that includes the nucleotide sequence of a target nucleic acid,such as, for example, the amplification product obtained by amplifying atarget nucleic acid or the cDNA produced upon reverse transcription ofan RNA target nucleic acid.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. I.e., if a nucleotide at agiven position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

“Specific hybridization” refers to the binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Those of skill in the art recognize thatrelaxing the stringency of the hybridization conditions allows sequencemismatches to be tolerated.

In particular embodiments, hybridizations are carried out understringent hybridization conditions. The phrase “stringent hybridizationconditions” generally refers to a temperature in a range from about 5°C. to about 20° C. or 25° C. below than the melting temperature (T_(m))for a specific sequence at a defined ionic strength and pH. As usedherein, the T_(m) is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half-dissociated intosingle strands. Methods for calculating the T_(m) of nucleic acids arewell known in the art (see, e.g., Berger and Kimmel (1987) METHODS INENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego:Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: ALABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory),both incorporated herein by reference). As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative FilterHybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The meltingtemperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the primer or probe and nature ofthe target nucleic acid (DNA, RNA, base composition, present in solutionor immobilized, and the like), as well as the concentration of salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol). The effects of these factors are wellknown and are discussed in standard references in the art. Illustrativestringent conditions suitable for achieving specific hybridization ofmost sequences are: a temperature of at least about 60° C. and a saltconcentration of about 0.2 molar at pH7.

The term “oligonucleotide” is used to refer to a nucleic acid that isrelatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides, most particularly, shorterthan 50 nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but primers are typically at least 7nucleotides long and, more typically range from 10 to 30 nucleotides, oreven more typically from 15 to 30 nucleotides, in length. Other primerscan be somewhat longer, e.g., 30 to 50 nucleotides long. In thiscontext, “primer length” refers to the portion of an oligonucleotide ornucleic acid that hybridizes to a complementary “target” sequence andprimes nucleotide synthesis. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with atemplate. The term “primer site” or “primer binding site” refers to thesegment of the target nucleic acid to which a primer hybridizes.

A primer is said to anneal to another nucleic acid if the primer, or aportion thereof, hybridizes to a nucleotide sequence within the nucleicacid. The statement that a primer hybridizes to a particular nucleotidesequence is not intended to imply that the primer hybridizes eithercompletely or exclusively to that nucleotide sequence.

The term “primer pair” refers to a set of primers including a 5′“upstream primer” or “forward primer” that hybridizes with thecomplement of the 5′ end of the DNA sequence to be amplified and a 3′“downstream primer” or “reverse primer” that hybridizes with the 3′ endof the sequence to be amplified. As will be recognized by those of skillin the art, the terms “upstream” and “downstream” or “forward” and“reverse” are not intended to be limiting, but rather provideillustrative orientation in particular embodiments.

A “probe” is a nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,generally through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target.Alternatively, however, the probe may be unlabeled, but may bedetectable by specific binding with a ligand that is labeled, eitherdirectly or indirectly. Probes can vary significantly in size.Generally, probes are at least 7 to 15 nucleotides in length. Otherprobes are at least 20, 30, or 40 nucleotides long. Still other probesare somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotideslong. Yet other probes are longer still, and are at least 100, 150, 200or more nucleotides long. Probes can also be of any length that iswithin any range bounded by any of the above values (e.g., 15-30nucleotides in length).

The primer or probe can be perfectly complementary to the target nucleicacid sequence or can be less than perfectly complementary. In certainembodiments, the primer has at least 65% identity to the complement ofthe target nucleic acid sequence over a sequence of at least 7nucleotides, more typically over a sequence in the range of 10-30nucleotides, and often over a sequence of at least 14-25 nucleotides,and more often has at least 75% identity, at least 85% identity, atleast 90% identity, or at least 95%, 96%, 97%. 98%, or 99% identity. Itwill be understood that certain bases (e.g., the 3′ base of a primer)are generally desirably perfectly complementary to corresponding basesof the target nucleic acid sequence. Primer and probes typically annealto the target sequence under stringent hybridization conditions.

“Amplification” encompasses any means by which at least a part of atleast one target nucleic acid is reproduced, typically in atemplate-dependent manner, including without limitation, a broad rangeof techniques for amplifying nucleic acid sequences, either linearly orexponentially. Illustrative means for performing an amplifying stepinclude ligase chain reaction (LCR), ligase detection reaction (LDR),ligation followed by Q-replicase amplification, PCR, primer extension,strand displacement amplification (SDA), hyperbranched stranddisplacement amplification, multiple displacement amplification (MDA),nucleic acid strand-based amplification (NASBA), two-step multiplexedamplifications, rolling circle amplification (RCA), and the like,including multiplex versions and combinations thereof, for example butnot limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR,LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), and thelike. Descriptions of such techniques can be found in, among othersources, Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach,Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book,Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996);The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press,Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993February; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451,Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCTPublication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162(1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press (1990);Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al.,Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development ofa Multiplex Ligation Detection Reaction DNA Typing Assay, SixthInternational Symposium on Human Identification, 1995 (available on theworld wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html-);LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene,2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi andSambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. AcidRes. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat.No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No.WO9803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally.

The term “qPCR” is used herein to refer to quantitative real-timepolymerase chain reaction (PCR), which is also known as “real-time PCR”or “kinetic polymerase chain reaction.”

A “reagent” refers broadly to any agent used in a reaction, other thanthe analyte (e.g., nucleic acid being analyzed). Illustrative reagentsfor a nucleic acid amplification reaction include, but are not limitedto, buffer, metal ions, polymerase, reverse transcriptase, primers,template nucleic acid, nucleotides, labels, dyes, nucleases, and thelike. Reagents for enzyme reactions include, for example, substrates,cofactors, buffer, metal ions, inhibitors, and activators.

“Hydrolysis probes” are generally described in U.S. Pat. No. 5,210,015,which is incorporated herein by reference in its entirety for itsdescription of hydrolysis probes. Hydrolysis probes take advantage ofthe 5′-nuclease activity present in the thermostable Taq polymeraseenzyme typically used in the PCR reaction (TagMan® probe technology,Applied Biosystems, Foster City Calif.). The hydrolysis probe is labeledwith a fluorescent detector dye such as fluorescin, and an acceptor dyeor quencher. In general, the fluorescent dye is covalently attached tothe 5′ end of the probe and the quencher is attached to the 3′ end ofthe probe, and when the probe is intact, the fluorescence of thedetector dye is quenched by fluorescence resonance energy transfer(FRET). The probe anneals downstream of one of the primers that definesone end of the target nucleic acid in a PCR reaction. Using thepolymerase activity of the Taq enzyme, amplification of the targetnucleic acid is directed by one primer that is upstream of the probe anda second primer that is downstream of the probe but anneals to theopposite strand of the target nucleic acid. As the upstream primer isextended, the Taq polymerase reaches the region where the labeled probeis annealed, recognizes the probe-template hybrid as a substrate, andhydrolyzes phosphodiester bonds of the probe. The hydrolysis reactionirrevocably releases the quenching effect of the quencher dye on thereporter dye, thus resulting in increasing detector fluorescence witheach successive PCR cycle. In particular, hydrolysis probes suitable foruse in the invention can be capable of detecting 8-mer or 9-mer motifsthat are common in the human and other genomes and/or transcriptomes andcan have a high T_(m) of about 70° C. enabled by the use of linkednucleic acid (LNA) analogs.

The term “label,” as used herein, refers to any atom or molecule thatcan be used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached, directly or indirectly, to anucleic acid or protein. Suitable labels that can be attached to probesinclude, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates.

The term “dye,” as used herein, generally refers to any organic orinorganic molecule that absorbs electromagnetic radiation at awavelength greater than or equal 340 nm.

The term “fluorescent dye,” as used herein, generally refers to any dyethat emits electromagnetic radiation of longer wavelength by afluorescent mechanism upon irradiation by a source of electromagneticradiation, such as a lamp, a photodiode, or a laser.

The “reverse transcriptase (RT) region of the polymerase (Pol) gene ofHepatitis B Virus (HBV)” is a region of the HBV Pol gene that encodesthe RT domain. More specifically, the HBV polymerase protein can bedivided into 4 domains (terminal protein, spacer, RT, ribonuclease H),and each of these can be numbered separately. As used herein, the HBV RTdomain starts with the highly conserved EDWGPCDEHG motif, contains 344amino acids, and, as an example of resistance against anti-HBV drugtherapies, the lamivudine-related resistance mutations are found atamino acid rtL180M and rtM204V/I (previously 552, 550, 539, or 549).

As used herein, the “target RT region” is a region of the HBV Pol genethat overlaps with the surface antigen (SA) gene.

HBV nucleotide numbers are numbered from nucleotide 1 of the circulargenome, which, by convention begins at an EcoRI cleavage site.

Amplification and DNA Sequencing for HBV Typing and ResistanceDetermination—In General

In particular embodiments, the invention includes an amplification andDNA sequencing method for detecting and analyzing the nucleotidesequence of a reverse transcriptase (RT) region of the polymerase (Pol)gene of Hepatitis B Virus (HBV). The method is carried out to determinethe HBV genotype or serotype of a product amplified from this regionand/or the presence or absence of one or more DNA sequencescharacteristic of an HBV mutation that confers resistance to a drug orvaccine. In preferred embodiments, both types of information are derivedfrom one amplification and sequencing assay. The method entailscontacting a nucleic acid sample with a primer pair specific for atarget RT region and carrying out an amplification reaction to produceand quantify an amplified product if the target RT region is present inthe sample. If an amplified product is obtained, its DNA sequence isthen determined and compared to one or more DNA sequences characteristicof an HBV genotype or serotype; and/or one or more DNA sequencescharacteristic of an HBV mutation that confers resistance to a drug orvaccine. The DNA sequences characteristic of the nine major HBVgenotypes (A-I) and four major serotypes (adr, adw, ayr, ayw) are wellknown (see Background section). The DNA sequence comparison can also becarried out to determine HBV subgenotype, as well as HBV genotype and/orserotype, including any genotype, subgenotype, or serotype for which DNAsequence information becomes available in the future. DNA sequencescharacteristic of mutations that confer resistance to a drug or vaccineare also well known (see Background section). In various embodiments,such mutations include those that confer resistance to lamivudine,adefovir, entecavir, telbivudine, tenofovir, or any combination thereof.Furthermore, the DNA sequence comparison can be carried out with DNAsequences that are identified in the future as conferring resistance toany available HBV drug or vaccine.

Amplification can be carried out using any suitable amplificationmethod, such as, e.g., polymerase chain reaction (PCR). In particularembodiments, the amplification reaction is a real-time amplificationreaction, such as real-time PCR (RT-PCT). Amplification methods aredescribed in greater detail below. DNA sequencing can also be carriedout using any of the various available DNA sequencing methods, such as,e.g., cycle DNA sequencing. DNA sequencing methods are described in moredetail below.

In certain embodiments, the method additionally includes determiningamount of amplified product produced and, optionally, diluting theamplified product prior to sequencing. The amplified product can bereadily quantified during real-time amplification by any of a variety ofavailable methods. If the concentration of the amplified product isappropriate for the selected DNA sequencing method, the amplifiedproduct can be sequenced without adjusting its concentration. However,if the concentration of the amplified product is not appropriate for theselected DNA sequencing method, the amplified product may beconcentrated or, more typically, diluted to a more appropriate level. Invarious embodiments, the degree of dilution is 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8 or falls within a range bounded by any of these values. Incertain embodiments, the method included diluting the amplified productby either 1:2 or 1:5 prior to sequencing. Quantitation of the amplifiedproduct thus offers the advantage that conditions can be adjusted toprovide better sequencing results than in the absence of quantitation.

The primer pair specific for a target RT region is selected to producean amplified product that includes one or more sites that vary amonggenotypes, subgenotypes, and/or serotypes and/or one or more sitesassociated with drug or vaccine resistance. In various embodiments, thisprimer pair is selected to produce an amplified product that includes atleast about: 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, or 1250 nucleotides, or anynumber of nucleotides falling within any range bounded by any of thesevalues, e.g., 1000-1050. In certain embodiments, this primer pair isselected to produce an amplified product includes a nucleotide sequencethat encodes an amino acid sequence including RT53 through RT256, asnumbered from the N-terminus of the RT domain. See FIG. 1. Primer designand illustrative primers are described further below.

In particular embodiments, the DNA sequence of the amplified product isdetermined using primers. The number of primers employed is generallysufficient to obtain DNA sequence for regions of interest. In certainembodiments, 4 primers are employed, namely:

-   -   a primer that anneals to the HBV genome 5′ of nucleotide 286,        which can be employed as a first forward primer;    -   a primer that anneals to the HBV genome 3′ of nucleotide 895,        which can be employed as a first reverse primer;    -   a primer that anneals to the HBV genome between nucleotide 377        and nucleotide 827, which can be employed as a second forward        primer; and    -   a primer that anneals to the HBV genome between nucleotide 377        and nucleotide 827, which can be employed as a second reverse        primer (in this case, this primer will anneal to the HBV genome        “downstream” of the second forward primer).

In some embodiments, one or more of the same primers can be used in theprimer pair specific for a target RT region and as a DNA sequencingprimer(s). In various embodiments, the primer pair specific for a targetRT region can include: (1) the first forward primer and the firstreverse primer, (2) the first forward primer and the second reverseprimer, (3) the second forward primer and the first reverse primer, (4)the second forward primer and the second reverse primer (in each ofthese instances, the primer designations refer to those introducedabove).

The methods described herein can be carried out to analyze nucleic acidsamples obtained from human patients, e.g., those known to have, orsuspected of having, an HBV infection. The information obtained (e.g.,HBV genotype or serotype and/or any HBV mutation associated with drugresistance and/or HBV vaccine escape) can, in certain embodiments, berecorded in a patient medical record, which can entail recording theinformation in a computer-readable medium. The patient medical recordcan be one that is maintained by a laboratory, physician's office, ahospital, a health maintenance organization, an insurance company, or ona personal medical record website.

In certain embodiments, the methods described above can additionallyinclude adjusting the treatment of a patient based on the informationobtained. Thus, the methods can include prescribing, initiating, and/oraltering drug therapy for HBV. For example, if the results indicate thepresence of an HBV strain having no known mutations associated with drugor vaccine resistance, a clinician can choose from among all availabledrug and vaccine treatments. However, if the results indicate thepresence of an HBV strain with a drug resistance mutation, the cliniciancould prescribe, initiate therapy with, or alter therapy to a differentdrug or a vaccine. Similarly, if the results indicate the presence of anHBV strain with a mutation associated with vaccine escape, the cliniciancould prescribe initiate therapy with, or alter therapy to anti-HBVimmunoglobulin (HBIG), or a drug.

Sample Nucleic Acids

Preparations of nucleic acids (“samples”) can be obtained frombiological sources and prepared using conventional methods known in theart. In particular, DNA or RNA useful in the methods described hereincan be extracted and/or amplified from any source, typically a samplefrom a human subject, such as a patient known to have, or suspected ofhaving, an HBV infection. Nucleic acids can be extracted or amplifiedfrom cells, bodily fluids (e.g., blood, a blood fraction, etc.), ortissue samples by any of a variety of standard techniques. Illustrativesamples include samples of plasma, serum, blood cells, stem cells, ortumors.

Nucleic acids of interest can be isolated using methods well known inthe art, with the choice of a specific method depending on the source,the nature of nucleic acid, and similar factors. The sample nucleicacids need not be in pure form, but are typically sufficiently pure toallow the amplification and DNA sequencing steps of the methodsdescribed herein to be performed.

Primers

Primers suitable for nucleic acid amplification are sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact length and composition of the primer willdepend on many factors, including, for example, temperature of theannealing reaction, source and composition of the primer, and where aprobe is employed, proximity of the probe annealing site to the primerannealing site and ratio of primer:probe concentration. For example,depending on the complexity of the target nucleic acid sequence, anoligonucleotide primer typically contains in the range of about 15 toabout 30 nucleotides, although it may contain more or fewer nucleotides.In various embodiments, the primer contains 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or a number ofnucleotides falling within any range bounded by any of these values. Theprimers should be sufficiently complementary to selectively anneal totheir respective strands and form stable duplexes. One skilled in theart knows how to select appropriate primer pairs to amplify the targetnucleic acid of interest.

For example, PCR primers can be designed by using any commerciallyavailable software or open source software, such as Primer3 (see, e.g.,Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386;www.broad.mit.edu/node/1060, and the like) or by accessing the Roche UPLwebsite. The amplicon sequences are input into the Primer3 program withthe UPL probe sequences in brackets to ensure that the Primer3 programwill design primers on either side of the bracketed probe sequence.

Primers may be prepared by any suitable method, including, for example,cloning and restriction of appropriate sequences or direct chemicalsynthesis by methods such as the phosphotriester method of Narang et al.(1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown etal. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite methodof Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; the solid supportmethod of U.S. Pat. No. 4,458,066 and the like, or can be provided froma commercial source.

Primers may be purified by using a Sephadex column (AmershamBiosciences, Inc., Piscataway, N.J.) or other methods known to thoseskilled in the art. Primer purification may improve the sensitivity ofthe methods of the invention.

In certain embodiments, the method described herein are carried outusing one or more of the following primers:

(1) A primer that anneals to the HBV genome between nucleotide 180 andnucleotide 204, which can be employed as a first forward primer for DNAsequencing and/or as the forward primer in a primer pair specific for atarget RT region, see, e.g., SEQ ID NO:1 in Table 1. This primer annealsto the third nucleotide of the codon for amino acid 17 through the thirdnucleotide of the codon for amino acid 25 of the RT region,corresponding to the second nucleotide of the codon for amino acid 9through the second nucleotide of the codon for amino acid 17 of thesurface antigen region.

(2) A primer that anneals to the HBV genome between nucleotide 1178 andnucleotide 1202, which can be employed as a first reverse primer for DNAsequencing and/or as the reverse primer in a primer pair specific for atarget RT region, see, e.g., SEQ ID NO:1 in Table 1. This primer annealsbeyond the last nucleotide of the RT region and the surface antigenregion; corresponding to the second nucleotide of the codon for aminoacid 6 through the second nucleotide of the codon for amino acid 14 ofthe RNaseH region of the polymerase gene).

(3) A primer that anneals to the HBV genome between nucleotide 420 andnucleotide 450, which can be employed as a second forward primer for DNAsequencing and/or as the forward primer in a primer pair specific for atarget RT region, see, e.g., SEQ ID NO:1 in Table 1. This primer annealsto the third nucleotide of the codon for amino acid 97 through the thirdnucleotide of the codon for amino acid 107 of the RT region,corresponding to the second nucleotide of the codon for amino acid 89through the second nucleotide of the codon for amino acid 99 of thesurface antigen region.

(4) A primer that anneals to the HBV genome between nucleotide 673 andnucleotide 704, which can be employed as a second reverse primer for DNAsequencing and/or as the reverse primer in a primer pair specific for atarget RT region, see, e.g., SEQ ID NO:1 in Table 1. This primer annealsto the first nucleotide of the codon for amino acid 182 through thesecond nucleotide of the codon for amino acid 192 of the RT region,corresponding to the third nucleotide of the codon for amino acid 173through the first nucleotide of the codon for amino acid 184 of thesurface antigen region).

TABLE 1 SEQ ID NO. Nucleotide Sequence SEQ ID NO: 1  5′TAGGACCCCTGCTCGTGTTACAGGC 3′ SEQ ID NO: 2  5′GTGGGGGTTGCGTCAGCAAACACTT 3′ SEQ ID NO: 3 5′TATGCCTCATCTTCTTGTTGGTTCTTCTGGA 3′ SEQ ID NO: 4 5′CGAACCACTGAACAAATGGCACTAGTAAACTG 3′

Primers useful in the methods described herein can comprise (include),consist essentially of, or consist of any of SEQ ID NOs:1-4. A primerthat “consists essentially of” a given sequence includes a sufficientportion of that sequence to have the basic and novel characteristic ofspecifically hybridizing to the complementary sequence under the assayconditions; however, such a primer may lack one or more nucleotides ofthe given sequence and/or may include additional nucleotides. In variousembodiments, a primer that “consists essentially of” a given sequence isat least 60%, 65%, 70%, 75%, 80%, 85% 90%, or 95% identical to thatsequence or has a degree of sequence identity that falls within a rangebounded by any of these values.

Probe

In certain embodiments, a probe is employed to determine the amount ofamplified product produced, e.g., for a sequencing assay. Probes aredesigned and employed to hybridize selectively to the target nucleicacids of interest. Probes can be perfectly complementary to the targetnucleic acid sequence or can be less than perfectly complementary. Asthose of skill in the art appreciate many of the considerations fordesigning, making, and purifying primers also apply to designing,making, and purifying probes. In particular, probes are typically in therange of about 15 to about 30 nucleotides, although they may containmore or fewer nucleotides. In various embodiments, the probe contains15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides, or a number of nucleotides falling within any range boundedby any of these values.

Probes useful in the methods described herein can hybridize to anysuitable sequence in the amplified product, although preferred probes donot hybridize to primer sequences. In an illustrative embodiment, theprobe has a nucleotide sequence including 5′ AGACTCGTGGTGGACTTCTCTCA3′(SEQ ID NO:5), which hybrizes to nucleotides 250-272 of the HBVgenome. This probe can be used, e.g., to detect an amplified productproduced using any of the following primer pairs:

-   -   SEQ ID NO:1 and SEQ ID NO:2;    -   SEQ ID NO:1 and SEQ ID NO:4;    -   SEQ ID NO:3 and SEQ ID NO:2; and    -   SEQ ID NO:3 and SEQ ID NO:4.

In certain embodiments, the probe is labeled. Where quantification ofamplified product is carried out in a real-time amplification method,the probe is conveniently labeled with one or more fluorescent label(s).In an illustrative embodiment, the probe includes a fluorophore and aquencher at either end of the molecule, e.g., 5′ Quasar 670AGACTCGTGGTGGACTTCTCTCA-BHQ2 3′. Such a probe can be used in afluorogenic nuclease assay for real-time quantification (see below).

The illustrative probe described herein can comprise (include), consistessentially of, or consist of SEQ ID NOs:5. A probe that “consistsessentially of” a given sequence includes a sufficient portion of thatsequence to have the basic and novel characteristic of specificallyhybridizing to the complementary sequence under the assay conditions;however, such a primer may lack one or more nucleotides of the givensequence and/or may include additional nucleotides. In variousembodiments, a priobe that “consists essentially of” a given sequence isat least 60%, 65%, 70%, 75%, 80%, 85% 90%, or 95% identical to thatsequence or has a degree of sequence identity that falls within a rangebounded by any of these values.

Amplification Methods

Any method of detection and/or quantification of nucleic acids can beused in the invention to detect amplification products. In oneembodiment, PCR (polymerase chain reaction) is used to amplify and/orquantify target nucleic acids. In other embodiments, other amplificationsystems or detection systems are used, including, e.g., systemsdescribed in U.S. Pat. No. 7,118,910 (which is incorporated herein byreference in its entirety for its description of amplification/detectionsystems) and Invader assays; PE BioSystems). In particular embodiments,real-time quantification methods are used. For example, “quantitativereal-time PCR” methods can be used to determine the quantity of a targetnucleic acid present in a sample by measuring the amount ofamplification product formed during the amplification process itself.

Fluorogenic nuclease assays are one specific example of a real-timequantification method that can be used successfully in the methodsdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan®method.” See U.S. Pat. No. 5,723,591; Heid et al., 1996, Real-timequantitative PCR Genome Res. 6:986-94, each incorporated herein byreference in their entireties for their descriptions of fluorogenicnuclease assays. It will be appreciated that while “TaqMan® probes” arethe most widely used for quantitative real-time PCR, the invention isnot limited to use of these probes; any suitable probe can be used.

Other detection/quantification methods that can be employed in themethods described herein include FRET and template extension reactions,molecular beacon detection, Scorpion detection, Invader detection, andpadlock probe detection.

DNA Sequencing Methods

The amplified product obtained from the amplification reaction can besequenced using any conventional sequencing method, includingtraditional Sanger sequencing (chain-terminator sequencing), cycle DNAsequencing (dye-terminator sequencing, and high-throughput methods, suchas Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS),Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing,SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing,Helioscope™ single molecule sequencing, Single Molecule SMRT™sequencing, Single Molecule real time (RNAP) sequencing, Nanopore DNAsequencing, and VisiGen Biotechnologies sequencing. The amplifiedproduct can also be sequenced by hybridization or mass spectroscopy.

In an illustrative embodiment, cycle DNA sequencing is employed tosequence the amplified product. See Example below. “Cycle DNAsequencing” refers to a modification of the traditional Sangersequencing method in which dideoxynucleotides are used in apolymerization reaction to create a nested set of DNA fragments withdideoxynucleotides at the 3′ terminus of each fragment. Cycle sequencingemploys a thermostable DNA polymerase that can be heated to atemperature that denatures double-stranded DNA and still retainactivity. The sequencing reaction can be repeated over and over again inthe same tube by heating the mixture to denature the DNA and thenallowing it to cool to anneal the primers and polymerize new strands.

Removal of Undesired Reaction Components

It will be appreciated that reactions involving complex mixtures ofnucleic acids in which a number of reactive steps are employed canresult in a variety of unincorporated reaction components, and thatremoval of such unincorporated reaction components, or reduction oftheir concentration, by any of a variety of clean-up procedures canimprove the efficiency and specificity of subsequently occurringreactions. In certain embodiments, the concentration of undesiredcomponents can be reduced by simple dilution. In particular embodiments,undesired components can be removed by a variety of enzymatic means.Alternatively, or in addition to the above-described methods, undesiredcomponents can be removed by purification.

Assay Performance

The assay methods described above were validated as described inExamples 2-7. Design validation testing was evaluated using two assayformats: A-m2000sp for sample preparation, m2000rt for PCR and PCRproduct quantitation and determination of dilution factor, ExoSAP-IT forpost-PCR clean-up, Perkin Elmer (PE) 9700 for cycle sequencing, sodiumacetate/ethanol for post-cycle sequencing clean-up, and AppliedBiosystems 3130xl for sequence analysis; B-m24sp for sample preparation,PE 9700 for PCR, agarose gel analysis for PCR product quantitation anddetermination of dilution factor, Qiagen column for post-PCR clean-up,PE 9700 for cycle sequencing, isopropanol for post-cycle sequencingclean-up, and AB 3130xl for sequence analysis.

Participants were trained using review of the package insert and theOperations Manuals and performed training runs that included runcontrols and HBV samples. Following training, each participant executedone run: format A or format B. Instrument format A runs containedNegative Control, Positive Control, 23 HBV-negative samples and 23HBV-positive samples (including some samples near the assay limit ofdetection). Instrument format B runs contained Negative Control,Positive Control, 8 HBV-negative samples, and 8 HBV-positive samples(including some samples near the assay limit of detection).

The expected results were assigned by the R&D group based on the targettypes, i.e., a positive sample was expected to provide valid HBVsequence, while a negative sample should have provided a PCR-negativeresult.

The validation established that the assay methods described above fullymeet the need for a reliable HBV sequencing assay for detection of HBVdrug resistance mutations and for determination of HBV genotype inplasma or serum, with sensitivity comparable to or better than otherequivalent commercially available assays. More specifically, certainembodiments of assay methods were tested and found the meet thefollowing acceptance criteria: The overall agreement rate between validsample results and the expected results shall be 95.0% or greater.HBV-negative samples should have PCR-negative results, and HBV-positivesamples (including some samples near the assay limit of detection)should have PCR-positive results and valid sequence. A valid sequence isdefined as one where the sequence is of high quality (>99.0% accurate atthe base level, covers the biological region of interest with 3-4 primersequences per sample, and is HBV because it lines up against the HBVreference sequence). A valid sequence is also sequence that is availablefor analysis using sequence analysis programs where genotype andpresence of mutations associated with drug resistance can be determined.In particular, the test results demonstrated that all negative specimensgave the expected negative results, while all the positive specimenswere sequenced with high quality sequence results.

In some embodiments, the assay method produces a sequencing result in95% or more of specimens containing HBV DNA at a concentration of atleast 1200, 1000, 800, 600, 400 or 200 IU/mL HBV DNA, e.g., when 0.5 mLis tested. See Examples 2 and 3. In particular embodiment, the assaymethod produces a sequencing result in 95% or more of specimenscontaining HBV DNA at a concentration falling within a range defined byany of these values (e.g., 200-400 IU/mL HBV DNA). In specificembodiments, this criterion is met when a perfect match exists betweenthe primers and probe and the specimen nucleotide sequence.

In certain embodiments, the assay method produces the same result morethan 95.0% of the time (where agreement is defined as ≧98% agreement atthe base-pair level in the HBV genome sequence 337-909), when used totest plasma (ACD-A, CPD, sodium EDTA, potassium EDTA), and serum.

In particular embodiments, the assay method produces the same result≧95.0% of the time (where agreement is defined as ≧98% agreement at thebase-pair level in the HBV genome sequence 337-909), when used to testanti-coagulated plasma (ACD-A, CPD, sodium EDTA, potassium EDTA), andserum. For example, certain embodiments of the assay methods describedherein were tested as follows: Fifty matched sets (potassium EDTA,sodium EDTA, CPD and ACD-D plasma and serum) were tested; 25 of themwere HBV-negative, and 25 of them were HBV positive. As compared to theserum control, 100% (95% CI 98.17%-100%) of the 200 pairs were matchedacceptably.

In some embodiments, the assay method is capable of detecting mixedbases greater than 50% of the time, when the two populations of basesare at equal concentration, for the mixture panel at the 1×105 IU/mLviral load level. See Example 4.

In certain embodiments, the assay method produces the same result ≧95.0%of the time (where agreement is defined as ≧98% agreement at thebase-pair level in the HBV genome sequence 337-909), when used to testspecimens containing potential cross-reactants and interferingsubstances found in patient samples. See Example 5.

In particular embodiments, the assay method has an analyticalspecificity of 99.5% or greater, calculated using the frequency ofrepeatedly reactive (i.e., false positive) results, when used to testspecimens from HBV-negative people. In variations of such embodiments,the analytical specificity is 100.0%. See Example 6.

In some embodiments, the assay method provides a total samplepreparation, amplification, sequence generation, and sequence analysistime of less than 72 hours per plate (24 results). In variations of suchembodiments, the total sample preparation, amplification, sequencegeneration, and sequence analysis time is less than 30 hours, e.g.,about 25 hours.

In certain embodiments, 95.0% or more of completed runs are valid whenjudged by run controls, e.g., when at least 50 runs are performed. Forexample, the valid rate for m2000sp/m2000rt runs was tested and found tobe 98.1% (52/53). The valid rate for m24sp/PE9700/Agarose Gel runs wastested and found to be 100.0% (16/16).

The embodiment described in detail in Example 1 meets all of theperformance criteria described above.

Kits

Kits according to the invention include one or more reagents useful forpracticing one or more assay methods of the invention. A kit generallyincludes a package with one or more containers holding the reagent(s)(e.g., primers and/or probe(s)), as one or more separate compositionsor, optionally, as admixture where the compatibility of the reagentswill allow. The kit can also include other material(s) that may bedesirable from a user standpoint, such as a buffer(s), a diluent(s), astandard(s), and/or any other material useful in sample processing,washing, or conducting any other step of the assay.

Kits according to the invention generally include instructions forcarrying out one or more of the methods described herein. Instructionsincluded in such kits can be affixed to packaging material or can beincluded as a package insert. While the instructions are typicallywritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to, electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), RF tags, and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

In certain embodiments, a kit for detecting and analyzing the nucleotidesequence of a reverse transcriptase (RT) region of the polymerase (Pol)gene of Hepatitis B Virus (HBV) includes:

a primer that anneals to the HBV genome 5′ of nucleotide 286;

a primer that anneals to the HBV genome 3′ of nucleotide 895; and

two primers that anneal to the HBV genome between nucleotide 377 andnucleotide 827.

In various embodiments, two of the primers in the kit define a target RTregion that includes at least about: 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, or1250 nucleotides, or any number of nucleotides falling within any rangebounded by any of these values, e.g., 1000-1050.

In particular embodiments, the kit can include one or more of thefollowing primers:

-   -   a primer that anneals to the HBV genome between nucleotide 180        and nucleotide 204, see, e.g., SEQ ID NO:1 in Table 1 (above);    -   a primer that anneals to the HBV genome between nucleotide 1178        and nucleotide 1202, see, e.g., SEQ ID NO:1 in Table 1 (above);    -   a primer that anneals to the HBV genome between nucleotide 420        and nucleotide 450, see, e.g., SEQ ID NO:1 in Table 1 (above);        and    -   a primer that anneals to the HBV genome between nucleotide 673        and nucleotide 704, see, e.g., SEQ ID NO:1 in Table 1 (above).

The kit can also include a probe described herein, optionally incombination with one or more of the primers described herein. Forexample, the kit can include an illustrative probe that has a nucleotidesequence including 5′ AGACTCGTGGTGGACTTCTCTCA 3′ (SEQ ID NO:5). Incertain embodiments, the probe is labeled, e.g., 5′ Quasar 670AGACTCGTGGTGGACTTCTCTCA-BHQ2 3′ (SEQ ID NO:5).

The primers and/or probe described herein can be package in any mannerthat facilitates their use in the methods described above. For example,a container can include a primer pair specific for a target RT region toproduce the amplified product, and a primer(s) for DNA sequencing can beprovided in a separate container or container(s).

EXAMPLES Example 1 HBV Sequencing Assay Biological Principles of theProcedure

The HBV Sequencing assay consists of two kits: the HBV SequencingControl Kit and the HBV Sequencing Reagent Kit.

The HBV Sequencing assay uses PCR to generate amplified product from thepolymerase region of the HBV DNA genome in clinical specimens. Thepresence of HBV amplification products is detected by measuring thefluorescence of the HBV probe that binds to the target during theextension/anneal step or by agarose gel electrophoresis. The amplifiedproduct is purified to remove unused primers and deoxynucleotidetriphosphates (dNTPs) using ExoSAP-IT® (United States Biochemical (USB)Corporation) or spin columns (QIAGEN). An optimal quantity of purifiedamplified product is subjected to Sanger-based cycle sequencing. Thecycle sequencing products are purified using sodium acetate/ethanol orisopropanol precipitation and resuspended in formamide prior toelectrophoresis on an Applied Biosystems (AB) Genetic Analyzer (3130,3130xl). DNA sequence data are analyzed using the programs: AB SegScape®and web-based programs, such as Evivar or Genafor. The resultinginformation can be used to help manage anti-HBV therapy.

Sample Preparation

The purpose of sample preparation is to extract and concentrate targetDNA, to make the target accessible for amplification, and to removepotential inhibitors of amplification from the extract. This process isaccomplished by the m2000sp, an automated sample preparation systemdesigned to use magnetic microparticle processes for the purification ofnucleic acids from samples. The Abbott m Sample Preparation System_(DNA)(4×24 Preps) reagents are used to lyse the virion, capture the nucleicacids and wash the particles to remove unbound sample components.Proteinase K is included in the lysis step to digest proteins associatedwith the nucleic acids.^(4,5) The bound nucleic acids are eluted andtransferred to a 96-deep well plate. The nucleic acids are then readyfor amplification. Sample preparation may also be performed using them24sp or manually.

Reagent Preparation and Reaction Plate Assembly

The HBV Sequencing amplification reagent components (HBV Pol PCR Mix,AmpliTaq Gold® Enzyme, Activation Reagent, and Uracil-DNA-Glycosylase(UNG)) are assembled by the operator. The Abbott m2000sp is used todispense the resulting master mix to the Abbott 96-Well Optical ReactionPlate along with nucleic acid samples prepared by the Abbott m2000sp.The plate is ready, after manual application of the Abbott OpticalAdhesive Cover, for transfer to the Abbott m2000rt. Alternatively, themastermix and samples can be manually dispensed, before amplification ina PE 9700 thermal cycler or equivalent.

Amplification

During the amplification/detection reaction, the target DNA is amplifiedby AmpliTaq Gold Enzyme in the presence of primers, Quasar-labeledprobe, deoxynucleotide triphosphates (dNTPs), deoxyuridine triphosphate(dUTP), UNG and magnesium chloride. First, the HBV primers anneal totheir respective targets and are extended by the polymerase. After adenaturation step in which the temperature of the reaction is raisedabove the melting point of the double-stranded DNA product, the newlycreated DNA strand is denatured from the target DNA. During each roundof thermal cycling, amplification products dissociate to single strandsat high temperature allowing primer annealing and extension as thetemperature is lowered. Exponential amplification of the product isachieved through repeated cycling between high and lower temperatures,resulting in a billion-fold or greater amplification of targetsequences. The target sequence for the HBV Sequencing assay includes aportion of the polymerase and overlapping Surface Antigen gene in theHBV genome. This region is specific for HBV and is highly conserved. Theprimers are designed to hybridize to this region with the fewestpossible mismatches among HBV genotypes A through H.

Detection

The presence of HBV amplification products is detected during theanneal/extension step by measuring the fluorescence of theQuasar-labeled HBV probe that binds to the target during theanneal/extension step. The HBV probe is a single-stranded DNAoligonucleotide consisting of a probe sequence with a fluorescent moietythat is covalently linked to the 5′ end of the probe and a quenchingmoiety that is covalently linked to the 3′ end of the probe. In theabsence of the HBV target sequence, probe fluorescence is quenched. Inthe presence of HBV, the HBV probe specifically binds to the target.This separates the fluorophore from the quencher, allowing fluorescentemission and detection. The amplification cycle at which fluorescentsignal is detected by the Abbott m2000rt is proportional to the log ofthe HBV DNA concentration present in the original sample. Alternatively,if an m2000rt was not used for amplification, detection of PCR productmay be performed using agarose gel analysis.

PCR Product Purification

The seal is removed from the Abbott 96-Well Optical Reaction Plate, andthe PCR reactions are purified to remove unused primers and reactioncomponents that may interfere with subsequent cycle sequencingreactions. Purification is achieved using ExoSAP-IT (USB), a reagentthat consists of a mixture of exonuclease I for degradation ofsingle-stranded primers, and Shrimp Alkaline Phosphatase for removal ofphosphate from dNTPs thus rendering them non-functional. Purificationcan also be achieved using a size separation spin column (QIAGEN).

Quantitation and Dilution

Quantitation of amplified product is achieved using the Cycle Number(CN) results from the m2000rt or by gel analysis. If amplicon isdetected, it can be diluted 1:2 or 1:5 to ensure that the optimalquantity of amplified product is added to cycle sequencing reactions.

Cycle Sequencing

Pol amplified product of each specimen is analyzed using four differentcycle sequencing reactions. Each type of cycle sequencing reactionscontains amplified product, a unique primer, and BigDye® Terminator v3.1(Applied Biosystems). Sequencing mixtures and purified amplifiedproducts are manually added to Abbott 96-Well Optical Reaction Platewhich is then sealed prior to cycle sequencing. During each thermalcycling step, the primer hybridizes to the amplified product and BigDyeTerminator extends the length of the complementary strands. Because aminor proportion of the substrate dNTPs are fluorescently-labeleddideoxynucleotides (ddNTPs), chain termination occurs at each positionin a minority of the growing chains. Each base-type of ddNTPs (A, C, G,T) is labeled with a different fluorophore. Chain termination results inthe generation of extension product, each one nucleotide longer than itspredecessor.⁶

Cycle Sequencing Purification

The seal is removed from the Abbott 96-Well Optical Reaction Plate, andthe cycle sequencing reactions are purified to remove labeled ddNTPs,unused primers, and other reaction components that may interfere withcapillary electrophoresis. Purification is achieved using sodiumacetate/ethanol or isopropanol precipitation. Purified DNA isresuspended in formamide.

Capillary Electrophoresis

The purified cycle sequencing product is analyzed using acapillary-based Genetic Analyzer. DNA products are separated on thebasis of their size with smaller products traveling faster than largerones. Because each ddNTP base-type is labeled with a unique fluorophore,the sequence of the DNA target can be determined by the order in whichthe fluorescently-labeled DNA products pass by the exciter/detector cellin the Genetic Analyzer.⁶

Data Analysis

Each specimen can have up to four different primer reads (samples). TheSeqScape program is used to assemble a contig from up to four differentreads and analyze the data. The resulting consensus sequence may beexported and analyzed using web-based analysis programs, such as Evivaror Genafor.

Reagents

Rare reagents include the five different oligonucleotides found in theOligo Mix and/or Sequencing Primers A-D as well as the thermostableAmpliTaq Gold.

HBV Sequencing Reagent Kit (List No. 3NO3-90)

(1) HBV Pol PCR Mix (3NO3Llbl) (1 vial, approximately 1.55 mL, for 48reactions), 19.447 mM Tris-HCl, pH 8.3, 97.235 mM KCl, 0.097 mM EDTA,0.389 mM each of dATP, dCTP, dGTP, dTTP, 0.194 mM dUTP, 0.486 uMHBVFP180-204 (5′ TAGGACCCCTGCTCGTGTTACAGGC 3′; SEQ ID NO:1), 0.486 uMHBVRP1202-1178 (5′ GTGGGGGTTGCGTCAGCAAACACTT 3′; SEQ ID NO:2), 0.778 uMQuasar-HBVPb250-272 (5′ Quasar/AGACTCGTGGTGGACTTCTCTCA/BHQ2 Quencher 3′;SEQ ID NO:5), 0.039 uM Rox dye, 0.085% Sodium Azide, 0.147% Proclin 950,molecular biology grade water.

(2) AmpliTaq Gold Enzyme (3NO3Tlbl) (1 vial, 0.078 mL), recombinantthemostable AmpliTaq Gold. 5 units/ul.

(3) Activation Reagent (3NO3Mlbl) 1 vial (0.778 mL), 38 mM MagnesiumChloride, 30 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.150% Proclin 950, 0.084%Sodium Azide, and molecular biology grade water.

(4) Uracil-DNA-Glycosylase (3NO3UNGlbl) (1 vial, 0.060 mL), recombinantUNG. 1 unit/ul.

(5) BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Mix(3NO3BDYElbl) (1 vial, 0.9 mL)

(6) HBV Sequencing Pol Primer A (3NO3Albl) (1 vial, approximately 0.13mL, for 48 reactions), 3.2 uM HBVFP180-204 primer (5′TAGGACCCCTGCTCGTGTTACAGGC 3′; SEQ ID NO:1) in Tris-EDTA buffer.

(7) HBV Sequencing Pol Primer B (3NO3BigDyelbl) (1 vial, approximately0.13 mL, for 48 reactions), 3.2 uM HBVFP420-450 primer (5′TATGCCTCATCTTCTTGTTGGTTCTTCTGGA 3′; SEQ ID NO:3) in Tris-EDTA buffer.

(8) HBV Sequencing Pol Primer C (3NO3Clbl) (1 vial, approximately 0.13mL, for 48 reactions), 3.2 uM HBVRP704-673 primer (5′CGAACCACTGAACAAATGGCACTAGTAAACTG 3′; SEQ ID NO:4) in Tris-EDTA buffer.

(9) HBV Sequencing Pol Primer D (3NO3Dlbl) (1 vial, approximately 0.13mL, for 48 reactions), 3.2 uM HBVRP1202-1178 primer (5′GTGGGGGTTGCGTCAGCAAACACTT 3′; SEQ ID NO:2) in Tris-EDTA buffer.

HBV Sequencing Control Kit (List No. 3NO3-80)

(1) Abbott HBV Sequencing Negative Control (4 vials, 1.3 mL per vial).Negative human plasma tested and found to be nonreactive for HBsAg, HIVRNA, anti-HIV-1/HIV-2, HBV DNA, HCV RNA, and anti-HCV by FDA licensedtests. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

(2) Abbott HBV Sequencing Positive Control (4 vials, 1.3 mL per vial).Heat-Inactivated HBV Virus Stock diluted to 4.477 log IU/mL in negativehuman plasma (when manufactured, the negative plasma was tested, andfound to be non-reactive for HBsAg, HIV RNA, anti-HIV-1/HIV-2, HBV DNA,HCV RNA, and anti-HCV by FDA licensed tests). Preservatives: 0.1%ProClin 300 and 0.15% ProClin 950.

Specimen Collection, Storage, and Transport to the Test Site

Specimen Collection and Storage

Human serum and plasma (ACD, EDTA, and CPD) specimens may be used withthe HBV Sequencing assay. Freshly drawn specimens (whole blood) can beheld at 2 to 30° C. for up to 6 hours prior to centrifugation. Aftercentrifugation, remove serum or plasma from cells. Serum or plasmaspecimens may be stored: at 15 to 30° C. for up to 24 hours; at 2 to 8°C. for up to 3 days; or at −10° C. or colder for longer term.

Multiple freeze-thaw cycles should be avoided. If frozen, thaw specimensat 15 to 30° C. or at 2 to 8° C. Once thawed, if specimens are not beingprocessed immediately, they can be stored at 2 to 8° C. for up to 6hours.

HBV Sequencing Assay Procedure

Work Areas

Use three dedicated areas within the laboratory for performing the HBVSequencing assay.

The Sample Preparation Area is dedicated to processing samples(specimens and HBV Sequencing Controls), and to adding processed samplesand controls to the Abbott 96-Well Optical Reaction Plate. After manualpreparation of the amplification master mix, it is distributed to thereaction plate. All reagents used in the Sample Preparation Area shouldremain in this dedicated area at all times. Laboratory coats, pipettes,pipette tips, and vortexers used in the Sample Preparation Area shouldremain in this area and not be moved to the Amplification Area. Do notbring amplification product into the Sample Preparation Area.

The Amplification Area is dedicated to PCR amplification. Laboratorycoats and equipment used in the Amplification Area should remain in thisarea and not be moved to the other areas.

The Sequencing Area is dedicated to gel electrophoresis, amplifiedproduct purification, cycle sequencing, and sequence analysis.Laboratory coats and equipment used in the Sequencing Area should remainin this area and not be moved to the other areas.

Sample Preparation Area

All specimen storage and preparation should take place in the dedicatedSample Preparation Area. Specimen preparation can be performed usingm2000sp or m24sp.

Specimen Preparation Performed Using m2000sp

1. A total of 48 samples can be processed in each run. A negativecontrol and a positive control must be included in each run, thereforeallowing a maximum of 46 specimens to be processed per run. Check samplevolume. Refer to the m2000sp Operations Manual and QUICK REFERENCE GUIDEFOR SAMPLE TUBE SIZES AND VOLUMES for recommended sample input volume.If frozen, thaw specimens at 15 to 30° C. or at 2 to 8° C. Once thawed,if specimens are not being processed immediately, store at 2 to 8° C.for up to 6 hours. Before use, vortex specimens three times for 2 to 3seconds. Ensure that bubbles or foam are not created. If found, removethem with a new sterile pipette tip for each tube. Specimens showingparticulate matter or turbidity should be clarified by centrifugation at2000 g for 5 minutes prior to testing. Aliquot each specimen into cleantubes or vials if necessary. Refer to the Abbott m2000sp OperationsManual for tube sizes. Avoid touching the inside of the cap when openingtubes.

2. Thaw assay controls at 15 to 30° C. or at 2 to 8° C. Once thawed, ifcontrols are not being processed immediately, store at 2 to 8° C. for upto 24 hours. Vortex each control three times for 2 to 3 seconds beforeuse. Ensure that bubbles or foaming are not created. If found, removethem with a new sterile pipette tip for each tube. Ensure that thecontents of the vials are at the bottom after vortexing by tapping thevials on the bench to bring liquid to the bottom of the vials.

3. Thaw amplification reagents at 15 to 30° C. or at 2 to 8° C. untilrequired for the amplification master mix procedure. This step can beinitiated before completion of the sample preparation procedure. Note:Do not vortex the amplification reagents. Once thawed, the amplificationreagents can be stored at 2 to 8° C. for up to 24 hours if not usedimmediately.

4. Open the Abbott Proteinase K reagent pack. Add 17.15 mL of MolecularBiology Grade water to a 50 mL polypropylene centrifuge tube. Pipet 2.45mL of Proteinase K into the container of water. Mix by gentle inversion10 to 15 times. Transfer the entire contents to a reagent vessel labeledwith the Proteinase K barcode label. Place the reagent vessel in reagentcarrier #1 location 2. NOTE: Use one bottle of Proteinase K solution andone set of mSample_(DNA) reagents to support up to 24 reactions. Use asecond set of Proteinase K and mSample_(DNA) reagents to support 25 to48 reactions, with the exception of the mMicroparticles. One bottle ofmMicroparticles will support up to 48 reactions.

5. Open the Abbott mSample Preparation pack. If crystals are observed inany of the reagent bottles upon opening, allow the reagents toequilibrate at room temperature until the crystals disappear. Do not usethe reagents until the crystals have dissolved.

6. Prepare the mWash2_(DNA) by adding 70 mL of USP Grade 190-200 ProofEthanol (95-100% Ethanol) to the mWash2_(DNA) bottle as described in theAbbott mSample Preparation System_(DNA) product information. Do not useethanol that contains denaturants.

7. Gently invert the Abbott mSample Preparation_(DNA) bottles to ensurea homogeneous solution and pour the contents into the appropriatereagent vessels per the Abbott m2000sp Operations Manual, OperatingInstructions.

8. Place the negative control, the positive control, and the patientspecimens into the m2000sp sample rack.

9. Place the 5 mL Reaction Vessels into the m2000sp 1 mL subsystemcarrier.

10. Load the carrier racks containing the Abbott mSamplePreparation_(DNA) reagents and Proteinase K, and the Abbott 96-Deep WellPlate, on the Abbott m2000sp worktable as described in the Abbottm2000sp Operations Manual.

11. From the Run Sample Extraction screen, select the appropriateapplication file. Initiate the sample extraction protocol as describedin the m2000sp Operations Manual, Operating Instruction. Followingsample scan, if samples are not labeled with barcodes, manually enterthe Sample IDs for those samples.

12. Sample eluates may be stored for up to 7 days at −10° C. or colder.

Sample Preparation Performed Using m24sp

13. A total of 18 samples can be processed in each run. A negativecontrol and a positive control must be included in each run, thereforeallowing a maximum of 16 specimens to be processed per run. Check samplevolume. Refer to the m24sp Operations Manual for recommended sampleinput volume. If frozen, thaw specimens at 15 to 30° C. or at 2 to 8° C.Once thawed, if specimens are not being processed immediately, store at2 to 8° C. for up to 6 hours. Before use, vortex specimens three timesfor 2 to 3 seconds. Ensure that bubbles or foam are not created. Iffound, remove them with a new sterile pipette tip for each tube.Specimens showing particulate matter or turbidity should be clarified bycentrifugation at 2000 g for 5 minutes prior to testing. Aliquot eachspecimen into clean tubes or vials if necessary. Refer to the Abbottm24sp Operations Manual for tube sizes. Avoid touching the inside of thecap when opening tubes.

14. Thaw assay controls at 15 to 30° C. or at 2 to 8° C. Once thawed, ifcontrols are not being processed immediately, store at 2 to 8° C. for upto 24 hours. Vortex each control three times for 2 to 3 seconds beforeuse. Ensure that bubbles or foaming are not created. If found, removethem with a new sterile pipette tip for each tube. Ensure that thecontents of the vials are at the bottom after vortexing by tapping thevials on the bench to bring liquid to the bottom of the vials.

15. Thaw amplification reagents at 15 to 30° C. or at 2 to 8° C. untilrequired for the amplification master mix procedure. This step can beinitiated before completion of the sample preparation procedure. Note:Do not vortex the amplification reagents. Once thawed, the amplificationreagents can be stored at 2 to 8° C. for up to 24 hours if not usedimmediately.

16. Open the Abbott Proteinase K reagent pack. Add 17.15 mL of MolecularBiology Grade water to a 50 mL polypropylene centrifuge tube. Pipet 2.45mL of Proteinase K into the container of water. Mix by gentle inversion10 to 15 times. Calculate the volume of Proteinase K solution requiredfor the m24sp run: (425 uL×number of samples) +1.0 mL for dead volume.Pipet the required volume into a Master Mix Tube (list number 04J71-80).

17. Open the Abbott mSample Preparation pack to check for crystals. Ifcrystals are observed in any of the reagent bottles, allow the reagentto equilibrate at room temperature until the crystals disappear. Do notuse the reagents until the crystals have dissolved.

18. Prepare the mWash2_(DNA) by adding 70 mL of USP Grade 190-200 ProofEthanol (95-100% Ethanol) to the mWash2_(DNA) bottle as described in theAbbott mSample Preparation System_(DNA) product information. Do not useethanol that contains denaturants. Invert the bottle to mix thecontents. If reusing the mSample Preparation SystemDNA reagents, markthe mWash2_(DNA) bottle to indicate that ethanol has already been added.

19. Load the Controls and patient specimens in the m24sp sample rack(s),starting with Position 3 of the IC Sample Rack. Place the Master MixTube containing the Proteinase K solution in the last position (16) ofSample Rack 2. CAUTION: Do NOT skip any positions in a sample rackexcept Position 1 and 2 of the IC Sample Rack. Load controls andspecimens into the sample racks in consecutive positions, starting withPosition 3 of the IC Sample Rack. Fill all positions in the IC SampleRack before loading specimens into Sample Rack 2. Insert specimen andcontrol tubes into sample racks carefully to avoid splashing. Ensurethat each tube is placed securely in the sample rack so that the bottomof the tube reaches the inside bottom of the rack. Load filled sampleracks on the worktable, starting with the IC Sample Rack in worktablePosition 1.

20. For each sample to be run, place one 5 mL Reaction Vessel into theSecondary Heat Block and one 1.5 mL Reaction Vessel in the m24sp TeMagSCarrier.

21. Load 1.5 mL Output Tubes or a 96 Deep-Well Plate onto the OutputLocation of the worktable. Load full racks of pipette tips in the DiTiCarriers and place them on the worktable. Place an empty tip rack and aclean deep-well plate on the Reuse Rack.

22. Remove the caps from the mSample Preparation SystemDNA reagentsexcept the mMicroparticlesDNA bottle. Store the caps on a clean,absorbent surface in the event that recapping is needed after the run.Place the uncapped reagent bottles in their designated positions in theReagent Carrier. Load the Reagent Carrier on the worktable.

23. Ensure all caps have been removed from specimens, controls, and theProteinase K solution Master Mix Tube prior to starting the m24sp run.Select an HBV Sequencing m24sp assay script (m24sp Database v 4.0 orhigher) appropriate for the desired output vessel:m24sp_HBVSeq_DNA_(—)0.5mL_Tube; m24sp_HBVSeq_DNA_(—)0.5mL_DWP. Initiatethe m24sp sample extraction protocol as described in the Abbott m24spOperations Manual, Operating Instructions section.

24. When prompted, vigorously mix the mMicroparticles_(DNA) to fullyresuspend them. Uncap and immediately place the mMicroparticlesDNAbottle in the Reagent Carrier and continue the run. Store the cap on aclean, absorbent surface in the event that recapping is needed after therun.

25. When m24sp sample preparation is finished and if not immediatelyproceeding with the Master Mix Addition Protocol in Package Insert, capthe 1.5 mL Output Tubes or cover the 96 Deep-Well Plate and store at 2to 30° C. for 4 hours, or at −10° C. or colder for up to 7 days.

26. At the end of each run, remove the remaining reagents from the m24spworktable. The Abbott mSample PreparationDNA reagents may be used amaximum of 3 times over 14 days for up to 24 reactions when storedtightly capped at 15 to 30° C. The Proteinase K solution Master Mix Tubeand any remaining solution in the tube must be discarded. Follow theappropriate laboratory guidelines for disposal. Any remaining ProteinaseK solution in the 50 mL polypropylene centrifuge tube (prepared in Step3 of the ASSAY PROTOCOL section) may be used two more times over 14 dayswhen stored tightly capped at 2 to 8° C.

Amplification Area

Switch on and initialize the Abbott m2000rt in the amplification area.The Abbott m2000rt requires 15 minutes to warm up. NOTE: Remove glovesbefore returning to the Sample Preparation Area.

Sample Preparation Area

NOTE: Change gloves before handling the amplification reagents.

27. After sample preparation is completed, manually assemble the HBVSequencing assay Master Mix. Thaw Amplification Reagents at 2 to 8° C.or at 15 to 30° C. Once thawed, reagents may be stored at 2 to 8° C. forup to 24 hours prior to use. Amplification reagents may be used up tothree times after freeze-thaw. Prior to opening the amplificationreagents, ensure that the contents are at the bottom of the vials bytapping the vials in an upright position on the bench and mix the HBVPol PCR Mix and Activation Reagent vials. Combine the following volumesof reagents in a sterile, 1.7 mL microcentrifuge tube:

Reagent Volume for 1 Reaction (μL) HBV Pol PCR Mix 25.711 UNG 0.500AmpliTaq Gold Enzyme 0.500 Activation Reagent 3.289 Note: If usingm2000sp for automated mastermix dispense, make five additionalreactions-worth to compensate for process loss. Vortex the Master Mix(i.e. combined 4 reagents in table above) for 3 to 5 seconds to mix.Transfer the entire contents of the microcentrifuge tube to a cleanm2000 Master Mix Tube.

28. Select the appropriate deep well plate from the Run Master MixAddition screen that matches the corresponding sample preparationextraction. Initiate the Abbott m2000sp Master Mix Addition protocol.Follow the instructions as described in the Abbott m2000sp OperationsManual, Operating Instructions section. Note: If setting-up the PCRplate manually, add 30 ul of mastermix to each well to be used and 20 ulof each purified sample. On m2000rt, under Sample Type, select Control.Then select “HBVSEQ_NEG” for the negative control and “HBVSEQ_POS” forthe positive control. Subsequently, under Sample Type, select Patient.Then manually enter the Sample IDs for those samples. The m2000rtprotocol (step 31) must be started within 90 minutes of assembling thePCR Master Mix (step 27).

29. Place the 96-Well Optical Reaction Plate into the Splash-FreeSupport Base for transfer to the thermal cycler.

30. Seal the Abbott 96-Well Optical Reaction Plate after the Abbottm2000sp instrument has completed addition of samples and master mixaccording to the Abbott m2000sp Operations Manual, OperatingInstructions section.

31. Export the completed 96-Well Optical Reaction Plate results to a CD.

Amplification Area

32. Place the Abbott 96-Well Optical Reaction Plate in the Abbottm2000rt instrument. Import the m2000sp test order via CD per the ImportOrder instructions in the Abbott m2000rt Operations Manual, OperatingInstructions section.

After loading the plate, select “Start” to start the run. The followingthermal cycling parameters will be executed.

Stage Amplification Parameters Number of Cycles 1 50° C. for 10 min 1 295° C. for 10 min 1 3 95° C. for 20 sec 45 65° C. for 45 sec 66° C. for2 min 4 72° C. for 10 min 1 5 4° C. hold 1 Note: At the end of stage 3,the following message appears: “6304: PCR data acquisition complete. Runcan be stopped at any time. Initiating hold.” Ignore this message untilstage 4 is complete. Confirm that the sample temperature has reached 4°C. before stopping the run. Note: Do not leave plate on hold for morethan 24 hours. The residual UNG activity may degrade the amplified DNA.Note: Thermal cycling may be performed using a PE 9700, or equivalent.If so, program the thermal cycling parameters (making sure that thereaction volume is set to 50 uL and the ramp speed is set to PE 9600mode), place the sealed plate into the cycler, apply a MicroAmp OpticalFilm Compression Pad on top of the plate, close the cycler lid and startthe program.

33. Remove the 96-Well Reaction Plate from the m2000rt and seal in azip-lock bag for further processing. The 96-Well Reaction Plate may bestored at −10° C. or colder for up to 7 days before purification (in theSequencing Area).

Sequencing Area

34. Gel electrophoresis is required only if PCR amplification was notperformed using m2000rt. To perform gel electrophoresis:

-   -   a. If frozen, thaw the DNA mass ladder and the purified samples.    -   b. Prepare a 1% agarose gel in 1×TAE buffer containing 0.5 ug/mL        of ethidium bromide. Ensure that the wells can hold 6 uL of        sample.    -   c. Pour sufficient 1×TAE buffer, containing 0.5 ug/mL of        ethidium bromide, to cover the gel.    -   d. Remove the gel comb.    -   e. In separate tubes, mix 5 uL of each sample and 1 uL gel        loading buffer.    -   f. Mix 1 uL low DNA mass ladder, 4 uL water, and 1 uL gel        loading buffer.    -   g. Load the lanes with as follows: lane 1 6 uL of the DNA        ladder, lanes 2-onwards 6 uL of each sample.

Fragment Amount of DNA (ng) Band Size (kb) 1 μL Ladder 1 2.0 50 2 1.2 303 0.8 20 4 0.4 10 5 0.2 5

-   -   h. Electrophorese at 10 V/cm until the blue dye has migrated at        least 5 cm from the wells.    -   i. Examine the gel with UV light.    -   j. Record the image using an exposure time that does not        saturate the film and shows the differences in intensity of the        mass ladder fragments.    -   k. Examine each sample result. If a major band is found between        Band 2 (1.2 kb) and Band 3 (0.8 Kb) of the DNA size marker, the        sample is considered successfully amplified. It is acceptable if        bands 4 and 5 are not visualized.

35. There are two preferred choices for purification of PCR products:ExoSAP-IT or QIAGEN QIAquick:

-   -   a. To purify using ExoSAP-IT,        -   (1) Program the PE 9700 thermal cycler or equivalent with            the following thermal cycling parameters (making sure that            the reaction volume is set to 50 uL and the ramp speed is            set to PE 9600 mode), place the sealed plate into the            cycler, apply a MicroAmp Optical Film Compression Pad on top            of the plate, close the cycler lid and start the program.

Stage Parameters Number of Cycles 1 37° C. for 15 min 1 2 80° C. for 15min 1

-   -   -   (2) On a new 96 well plate, add 4 ul of ExoSAP-IT into as as            many wells as samples to be processed.        -   (3) Add 10 ul of each PCR product to its own well containing            ExoSAP-IT.        -   (4) Seal the plate before vortexing it for 3 to 5 seconds.        -   (5) Centrifuge the plate for 5 to 10 seconds at 2,000 xg.        -   (6) Place the plate into thermal cycler, apply a MicroAmp            Optical Film Compression Pad on top of the plate, close the            cycler lid and start the program.

    -   b. To purify using QIAGEN QIAquick,        -   (1) Add 250 uL of Buffer PB to each sample.        -   (2) Transfer each mixture to a spin column. Place each spin            column into a collection tube.        -   (3) Centrifuge for 1 min. at 13,000 rpm.        -   (4) Discard waste from each collection tube and place the            spin column back into the same collection tube.        -   (5) Add 750 uL of Buffer PE to each spin column. (If a new            bottle of Buffer PE is used, the PE Buffer concentrate must            be mixed with 100% ethanol prior to use. Add 220 mL 100%            ethanol to the bottle contents. Cap the bottle and invert it            8 times to mix.) Place each spin column into a collection            tube.        -   (6) Centrifuge for 1 min. at 13,000 rpm.        -   (7) Transfer each spin column into a labeled 1.5 mL or 1.7            mL microcentrifuge tube.        -   (8) Add 50 ul buffer PE to each tube.        -   (9) Centrifuge for 1 min. at 13,000 rpm.        -   (10) Discard spin columns and then cap the tubes.

36. After purification, process the samples immediately (ExoSAP-IT andQiagen) or store at −10° C. or colder for up to 7 days (Qiagen only).

37. To determine if samples require dilution, refer to the“Interpretation” column of the m2000rt Results log or list. If theInterpretation reads “Not detected See Package Insert,” the sample didnot amplify appropriately and therefore should not be taken forward inthe assay process. If the Interpretation reads “Dilute 1:2”, then add 10uL water (Ultrapure) to 10 uL of the sample. If the Interpretation reads“Dilute 1:5”, then add 40 uL water (Ultrapure) to 10 uL of the sample.After all samples have been appropriately diluted, vortex for 3 to 5seconds using a vortex mixer set to 1,700 rpm. Note: If PCR wasperformed using the PE 9700, or equivalent, and subsequent gelelectrophoresis, then examine the presence of a major PCR product bandbetween 1.2 and 0.8 kb (i.e. band 2 and band 3 from the top of the DNAmass ladder). Other minor band(s) of less intensity are acceptable. Ifthe major PCR product band intensity is ≧20 ng relative to the DNA massladder, dilute the sample 1:10. If a band is visible, but of <20 ngintensity, high quality sequence may not be obtained.

38. To perform cycle sequencing, do the following

-   -   a. Thaw Sequencing Primers at 2 to 8° C. or at 15 to 30° C. Once        thawed, primers may be stored at 2 to 8° C. for up to 24 hours        prior to use. Sequencing Primers may be used up to three times        after freeze-thaw.    -   b. Prior to opening the Sequencing Primer bottles, mix them and        then ensure that the contents are at the bottom of the vials by        tapping the vials in an upright position on the bench.    -   c. Combine the following volumes of reagents in a sterile, 1.7        mL microcentrifuge tube.

Component Volume Needed for 1 Sample (uL) BigDye Terminator v3.1 Cycle4.0 Sequencing RR-100 Primer A, B, C, or D 2.0 Total Volume 6.0

NOTE: Make additional reactions to compensate for pipetting loss.

39. Use the following diagram to aid in the addition of cycle sequencingreaction mixtures to a 96-well reaction plate (for 96 reactions).

1 2 3 4 5 6 7 8 9 10 11 12 A A A A A A A A A A A A A B B B B B B B B B BB B B C C C C C C C C C C C C C D D D D D D D D D D D D D E A A A A A AA A A A A A F B B B B B B B B B B B B G C C C C C C C C C C C C H D D DD D D D D D D D D

Pipette 6.0 uL of cycle sequencing Master Mix A into the appropriatenumber of wells in rows A and E. Pipette 6.0 uL of cycle sequencingMaster Mix B into the appropriate number of wells in rows B and F.Pipette 6.0 uL of cycle sequencing Master Mix C into the appropriatenumber of wells in rows C and G. Pipette 6.0 uL of cycle sequencingMaster Mix D into the appropriate number of wells in rows D and H.

40. Use the following diagram to aid in the addition of samples to a96-well reaction plate (for 96 reactions).

1 2 3 4 5 6 7 8 9 10 11 12 A PC S2 S4 S6 S8 S10 S12 S14 S16 S18 S20 S22B PC S2 S4 S6 S8 S10 S12 S14 S16 S18 S20 S22 C PC S2 S4 S6 S8 S10 S12S14 S16 S18 S20 S22 D PC S2 S4 S6 S8 S10 S12 S14 S16 S18 S20 S22 E S1 S3S5 S7 S9 S11 S13 S15 S17 S19 S21 S23 F S1 S3 S5 S7 S9 S11 S13 S15 S17S19 S21 S23 G S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 S21 S23 H S1 S3 S5 S7S9 S11 S13 S15 S17 S19 S21 S23

Pipette 4.0 uL of the Positive Control into wells A1, B1, C1, and D1.Pipette 4.0 uL of the first sample (S1) into wells E1, F1, G1, and H1.Continue adding 4.0 uL of the samples to the remaining columns accordingto the plate diagram Note: the Negative Control and any samples that didnot amplify should not be taken through cycle sequencing; only thePositive Control and reactive samples should be sequenced.

41. Seal the 96-Well PCR Reaction Plate with an optical adhesive cover.

42. Program and run the PE 9700 thermal cycler or equivalent as shownbelow (ensure that 9600 emulation mode is used during cycle sequencing;the alternative, Max, will result in poor quality sequencing data).

Cycle Sequencing Number of Stage Parameters Cycles 1 96° C. for 1 min 12 96° C. for 10 sec 25 50° C. for 5 sec 60° C. for 4 min 3 4° C. hold 1

43. Do not store the samples at 4° C. on the thermal cycler for longerthan 24 hours. Samples can be stored at −10° C. or colder for up tothree days.

44. There are two preferred choices to to purify cycle sequencingproducts: sodium acetate-ethanol precipitation and isopropanolprecipitation.

-   -   a. To purify using sodium acetate-ethanol precipitation        -   (1) Add 26 uL of freshly prepared ethanol/sodium acetate            solution to each well (to make this solution, for each            reaction combine 1 uL of 3M sodium acetate, pH 5.2, and 25            uL of 100% ethanol).        -   (2) Seal the plate with an optical adhesive cover.        -   (3) Vortex the plate for 1 minute.        -   (4) Centrifuge the plate for 20 minutes at 2,000×g.        -   (5) As soon as the centrifuge stops, carefully remove the            optical adhesive cover without disturbing the pellets.        -   (6) Immediately place an absorbent paper towel on top of the            plate and invert it.        -   (7) Place the plate in the centrifuge in the inverted            position, on top of paper towel, and centrifuge for 1 minute            at 150×g.        -   (8) Add 75 uL 70% ethanol to each well.        -   (9) Centrifuge the plate for 5 minutes at 2,000×g.        -   (10) Immediately place an absorbent paper towel on top of            the plate and invert it.        -   (11) Place the plate in the centrifuge in the inverted            position, on top of paper towel, and centrifuge for 1 minute            at 150×g.        -   (12) Analyze immediately or seal the plate with an optical            adhesive cover and store at −10′C or colder in the dark for            up to 7 days.    -   b. To purify using isopropanol precipitation        -   (1) Add 40 uL of freshly prepared 75% isopropanol solution            to each well (to make this solution, for each reaction            combine 30 uL of 100% isopropanol and 10 uL of deionized            water).        -   (2) Seal the plate with an optical adhesive cover.        -   (3) Vortex the plate for 1 minute.        -   (4) Centrifuge the plate for 45 minutes at 1,700×g.        -   (5) As soon as the centrifuge stops, carefully remove the            optical adhesive cover without disturbing the pellets.        -   (6) Immediately place an absorbent paper towel on top of the            plate and invert it.        -   (7) Place the plate in the centrifuge in the inverted            position, on top of paper towel, and centrifuge for 1 minute            at 700×g.        -   (8) When the centrifuge stops and drying is complete,            analyze the plate immediately or seal the plate and store it            at −10° C. or colder in the dark for up to 7 days

45. To perform electrophoresis on the AB Genetic Analyzer 3130/3130xlperform the following. Note: These instructions assume that the ABGenetic Analyzer 3130/3130xl computer is loaded with SeqScape software,version 2.5 or later, and has the “HBV_SEQv2” project template,described below, already entered. The project template file has beenprovided on the HBV Sequencing Application CD-ROM and can be importeddirectly into SeqScape. Alternatively, the project template may becreated manually by entering the parameters listed below (see SeqScapeUser Guide version 2.5 or later, AB Part Number 4359442). Note: Thetemplates assume the use of POP7 and Big Dye v3.1.

Protocol/Other Tab Type of Setting Setting Analysis Protocol GeneralName HBV_SEQ Basecalling Basecaller KB.bcp DyeSet/PrimerKB_3130_POP7_BDTv3.mob Processed Data True Profile Quality Threshold Donot assign N's to Basecalls Mixed Bases Mixed Bases Select. Use MixedBase Identification Call IUB if 2nd highest peak is ≧30% Clear Range Usequality values Select. Remove bases from the ends until fewer than 4bases out of 20 have QVs < 20 Use reference trimming Select. FilterMaximum Mixed Bases 20.0 (%) Maximum Ns (%) 10.0 Minimum Clear Length 50(bp) Minimum Sample 20 Score Analysis Defaults General Analysis DefaultsHBV_SEQ Name Project Gap Penalty 30.0 Extension Penalty  1.0 Couple theamino acid Select alignment to the nucleotide alignment Specimen GapPenalty 22.5 Extension Penalty  8.5 # Library Matches 20 BasecallSamples Select Sample Analysis Protocol HBV_SEQ Always use this Selectanalysis protocol Display Settings DefaultDisplaySetting Default SelectReference Data General Reference Data Group HBV_SEQv2 Group Name CodonTable Standard ROI Layer Reference: 1-3221 National Center forBiotechnology Information (NCBI) reference genotype A: X70185 Layer DrugResistance 130-1161 w/Translation Layer Validity Range 337-909w/Translation NT Variants Layer Position Reference Variant DescriptionDrug Resistance 238 T A 238A 238 T G 238G 506 T C 506C 517 G C 517C 517G T 517T 538 T A 538A 539 T G 539G 540 G C 540C 540 G T 540T 541 G A541A 541 G T 541T 542 C T 542T 550 A C 550C 550 A G 550G 550 A T 550T551 C G 551G 551 C T 551T 552 T G 552G 580 G A 580A 604 A G 604G 604 A T604T 605 G T 605T 610 A G 610G 610 A T 610T 611 T C 611C 611 T G 611G612 G A 612A 612 G C 612C 612 G T 612T 641 T C 641C 643 C T 643T 644 A C644C 697 A G 697G 707 A C 707C 748 A C 748C 748 A G 748G 750 G A 750A750 G C 750C 750 G T 750T AA Variants Layer AA position ReferenceVariant Description Drug Resistance 80 L I 80I 80 L V 80V 169 I T 169T173 V L 173L 180 L C 180C 180 L M 180M 181 A S 181S 181 A T 181T 181 A V181V 184 T A 184A 184 T C 184C 184 T F 184F 184 T G 184G 184 T I 184I184 T L 184L 184 T M 184M 184 T S 184S 194 A T 194T 202 S C 202C 202 S G202G 202 S I 202I 204 M I 204I 204 M S 204S 204 M V 204V 214 V A 214A215 Q S 215S 233 I V 233V 236 N T 236T 250 M I 250I 250 M L 250L 250 M V250V Variant Style Variant Settings NT Variants: Known change base -Blue Known insertion - Red Known deletion - Black Unknown change base -Green Unknown insertion - Magenta Unknown deletion - Yellow Crucialposition - Blue AA Variants Known change residue - Drug ResistantVariant Know insertion - Red Known deletion - Black Unknown changeresidue - Green Unknown insertion - Magenta Unknown deletion - YellowSilent mutation - Red Project HBV_SEQv2 Reference Data Group HBV_SEQv2Templates Analysis Defaults HBV_SEQ Display SettingsDefaultDisplaySettings for SeqScape v 2.0

-   -   a1. Power on the monitor. Power on the computer. In the Log on        to Windows dialog box: enter the user name and password, click        OK. Press the on/off button on the front of the sequencer (make        sure that the oven door is closed and locked and the instrument        doors are shut before turning on the instrument). Ensure that        the green status light is on and not flashing before proceeding.        If the green status light does not come on, start the Data        Collection software and view the log. Ensure that the daily,        weekly, monthly, and as-needed genetic analyzer instrument        maintenance tasks have been completed. Ensure that the 50 cm        capillary array is on the instrument (each capillary is designed        to support a minimum of 100 runs). Replace the POP7 polymer        bottle if the bottle has been on the instrument for longer than        7 days. Replace 1× Sequencing Buffer (before each run) up to the        respective fill lines in the Anode (position 1 in the tray) and        Cathode (next to the POP7 bottle) reservoirs. Replenish the        water reservoirs (positions 2 and 4 in the tray) using deionized        water.

Position 2 Position 4 Water Water Position 1 Position 3 1X SequencingBuffer Empty

-   -   b1. Remove bubbles in the lines, if present.    -   c1. Select the Data Collection icon on the computer desktop to        start 3130/3130xl Data Collection v3.0. As each application        activates, the red circles (off) change to yellow triangles        (activating)>, and then to green squares (on) when they are        fully functional.    -   d1. In Data Collection, click+to expand the subfolders in the        left tree pane.    -   e1. In the tree pane of the Data Collection software, click        Results Group. Click New. Under the General tab, name the        Results Group “HBV_SEQ”, add a Results Group Owner and, if        desired, a Results Group Comment. Under the Destination tab,        provide the Root Destination where the data will be stored.        Under Naming tab, under Name Delimiter, select the—delimiter.        Click OK. The HBV_SEQ Results Group can be used with all        subsequent runs.    -   f1. In the tree pane of the Data Collection software, click        Plate Manager.    -   g1. Click New to display the New Plate Dialog box.    -   h1. Type a name for the plate.    -   i1. Type a description for the plate (optional).    -   j1. Select SeqScape_YourInstrumentName in the Application        drop-down list.    -   k1. Select 96-well in the Plate Type drop-down list.    -   l1. Type a name for the owner and operator.    -   m1. Click OK to open the SeqScape Plate Editor.    -   n1. In the Sample Name column, enter a sample name for each        sample. Use the format: xxxxxxxxxx-A, xxxxxxxxxx-B,        xxxxxxxxxx-C, xxxxxxxxxx-D to designate each specimen being        sequenced by each sequencing primer.    -   o1. The Priority column can be manipulated to alter the priority        in which a sample is processed. A priority of 100 is assigned        automatically. Use a lower priority number to decrease a        sample's priority.    -   p1. In the Comments column, enter any comments for the sample.    -   q1. In the Project column, select Create New Project. Once the        Create New Project window opens, type the name of the project,        and select Project Template (HBV_SEQv2). Click OK. The selected        Project Template will appear in the Project Template column.    -   r1. In the Specimen column, create New Specimen ID for each        specimen (a specimen consists of four samples). Use the format        xxxxxxxxxx for the specimen name.    -   s1. In the Results Group 1 column, select the HBV_SEQ Results        Group.    -   t1. In the Instrument Protocol 1 column, select New to open the        Protocol Editor. Under Name, enter HBV_SEQ. Under Run Module,        select BDx_StdSeq50_POP7_(—)1. Under Dye Set, select Z_BigDyeV3.        Click OK. This instrument protocol can be used with all        subsequent runs.    -   u1. In the Analysis Protocol 1 column, select New. Create the        Analysis Protocol per the following table. The HBV_SEQ Analysis        Protocol can be used with all subsequent runs.

Tab Type of Setting Setting General Name HBV_SEQ Basecalling BasecallerKB.bcp DyeSet/Primer KB_3130_POP7_BDTv3.mob Processed Data True ProfileQuality Threshold Do not assign N's to Basecalls Mixed Bases Mixed BasesSelect. Use Mixed Base Identification Call IUB if 2nd highest peakis >30% Clear Range Use quality values Select. Remove bases from theends until fewer than 4 bases out of 20 have QVs < 20 Use referenceSelect. trimming Filter Maximum Mixed 20.0 Bases (%) Maximum Ns (%) 10.0Minimum Clear 50 Length (bp) Minimum Sample 20 Score

-   -   v1. Click OK to save then close the plate record. Note: After        clicking OK within the SeqScape Plate Editor, the completed        plate record is stored in the plate manager database. The plate        record can be searched for, edited, exported, or deleted in the        Plate Manager.    -   w1. If present, remove the seal from the plate to be analyzed.    -   x1. Add 20 uL Hi-Di Formamide to each sample-containing well.        The plate is stable for up at 35 hours at room temperature when        protected from light.    -   y1. Seal the plate with an optical adhesive cover.    -   z1. Vortex for 10 to 15 seconds at 1,700 rpm.    -   a2. Centrifuge the plate for 30-40 s at 2,000×g.    -   b2. Remove the optical adhesive cover, and lay a septa flat on        the plate.    -   c2. Align the holes in the septa strip with the wells of the        plate then firmly press downward onto the plate.    -   d2. Place the sample plate into the base plate.    -   e2. Snap the plate retainer onto the plate and plate base.    -   f2. Verify that the holes of the plate retainer and the septa        are aligned. If not, re-assemble the plate assembly.    -   g2. Press the Tray button and wait for the autosampler to stop        at the forward position.    -   h2. Open the front doors.    -   i2. Place the plate assembly on the autosampler in position A        or B. Note: There is only one orientation for the plate, with        the notched end of the plate base away from the operator.    -   j2. Ensure the plate assembly fits flat in the autosampler.        Failure to do so may allow the capillary tips to lift the plate        assembly off of the autosampler.    -   k2. Close the instrument doors.    -   l2. In the tree pane of the Data Collection software, click GA        Instruments >ga3130xl>Run Scheduler >Plate View.    -   m2. Click Find All.    -   n2. Select the plate record to be run.    -   o2. Click the plate position indicator that matches the plate to        be linked. The plate map color will change from yellow to green        when it is successfully linked.    -   p2. In the toolbar of the Data Collection software window, click        the green arrow to begin the run.    -   q2. The processing plates dialog box opens. Click OK.

30. Data Analysis:

-   -   a. After the run has completed, open SeqScape.    -   b. Select File, New Project. Give the project the same name as        when setting-up the Data Collection plate record.    -   c. In the New Project box, type the first few letters of the        project template, HBV_SEQv2. Click New.    -   d. Click File, Import Samples To Project. Find the sequencing        files. Click “Use sample name” and then select file(s). Click        Auto Add. Click OK.    -   e. Click the green arrow in the tool bar to initiate analysis.    -   f. After completion of analysis, select the run (not individual        specimens) within the Project Navigator.    -   g. For the Positive Control, check the following:        -   (1) In the Active Layer menu, select Layer Drug Resistance.            Under Analysis, click Report Manager, then click Specimen            Statistics Report. Verify that the number of samples is 3 or            greater and the Range on Reference covers nucleotides            337:909.        -   (2) In the Active Layer menu, select Layer Drug Resistance.            Under Analysis, Report Manager, click AA Variants Report.            Verify that the Positive Control contains no clinically            relevant amino acid variants at RT amino acid positions            associated with drug resistance (in the description column            of the SeqScape AA Variants Report).        -   (3) If a clinically relevant mutation is detected, the            accuracy of this determination may be checked by clicking on            the NT Change column of the AA Variants Report and, in turn,            clicking on the Base Change of the Mutations Report. To            check the electropherograms of the specimen in question,            click on the triangle before the Specimen Name within the            Project View. The nucleotide base may be edited if desired            (place the cursor on the base in question, press delete,            then type in the correct sequence) and the edits are            documented in the Audit Trail Report.        -   (4) If these criteria are not met (once review and editing            are complete), consider repeating the run.    -   h. For each specimen, check the following:        -   (1) In the Active Layer menu, select Layer Drug Resistance.            Under Analysis, click Report Manager, then click Specimen            Statistics Report. Verify that the number of samples is 3 or            greater and the Range on Reference covers nucleotides            337:909.        -   (2) In the Active Layer menu bar, selectLayer Drug            Resistance for analysis of drug resistance mutations. Under            Analysis, Report Manager, click AA Variants Report. Check to            determine if the specimen contains clinically relevant amino            acid variant/s listed in the Description column. If a            clinically relevant mutation is detected, the accuracy of            this determination may be checked by clicking on the NT            Change column of the AA Variants Report and, in turn,            clicking on the Base Change of the Mutations Report. To            check the electropherograms of the specimen in question,            click on the triangle before the Specimen Name within the            Project View. The nucleotide base may be edited if desired            (place the cursor on the base in question, press delete,            then type in the correct sequence) and the edits are            documented in the Audit Trail Report.    -   h. To export the specimen consensus sequence(s), perform the        following:        -   (1) To export an entire run containing multiple consensus            sequences, open File, select the whole file, click Export.        -   (2) To export an individual consensus sequence, open File,            select the sequence, click Export. Note: While open, reports            such as Specimen Statistics, AA Variants, or Audit Trail            Report, may be exported using the same methodology.    -   i. The specimen consensus sequence(s) may be sent to        Evivar-SeqHepB (www.seqhepb.com), or (www.genafor.org, then        select geno2pheno [hbv]) for clinical interpretation.

Post Processing Procedures

1. Store samples (unprocessed, post-PCR, post-purification, etc.) asrecommended in the procedure.

2. To minimize health hazards, and the potential spread of amplifiedproduct, ensure that waste materials (gloves, tips, etc.) are disposedof in sealed bags that are then autoclaved.

Quality Control Procedures

Abbott m2000rt Optical Calibration

Refer to the Calibration Procedures section in the Abbott m2000rtOperations Manual for a detailed description of how to perform an Abbottm2000rt Optical Calibration. Optical calibration of the Abbott m2000rtinstrument ensures the accurate measurement and discrimination of dyefluorescence during the HBV Sequencing assay. The following Abbottm2000rt Optical Calibration Plates are used to calibrate the Abbottm2000rtinstrument for the HBV Sequencing assay: Cy5™ Plate (Cyanine) andROX™ Plate (Carboxy-X-rhodamine). Additionally, please refer to AppliedBiosystems 3130/3130xl Genetic Analyzer Manuals for information on howto maintain and calibrate these instruments.

Negative and Positive Controls

A negative control and a positive control are included in each run toevaluate run validity. An error is displayed when a control result isout of range. Refer to the Abbott m2000rt Operations Manual for anexplanation of the corrective actions for the error code. If negative orpositive controls are out of range, all of the specimens and controlsfrom that run should be reprocessed, beginning with sample preparation.The presence of HBV should not be detected in the negative control. HBVdetected in the negative control is indicative of contamination by othersamples or by amplified product introduced during sample preparation orduring preparation of the Abbott 96-Well Optical Reaction Plate. Toavoid contamination, clean the Abbott m2000sp and m2000rt instrumentsand repeat the sample processing for controls and specimens followingthe Procedural Precautions. If negative controls are persistentlyreactive, assume that the laboratory is contaminated.

After completion of PCR, and optional gel electrophoresis, only thePositive Control should be processed further. The Positive Control helpsdetermine whether cycle sequencing and capillary electrophoresis wereperformed correctly. If the Positive Control is invalid, considerrepeating cycle sequencing and/or capillary electrophoresis. The assayis standardized against the World Health Organization (WHO)International Standard for Hepatitis B Virus DNA (NIBSC Code 97/746).⁷

Results

Two types of results are produced:

Post-PCR. This data is used to determine if the sample was amplifiedcorrectly, and if so, how the amplified product needs to be dilutedprior to cycle sequencing. Follow the m2000 report, ResultsInterpretation, to determine whether to dilute 1:2 or 1:5. Samplesdesignated as Not detected See Package Insert were not amplifiedappropriately and should not be processed further. Alternatively, if anm2000rt was not used, amplification success data, and associateddilution, can be obtained using gel electrophoresis.

Post-Sequencing. Nucleotide sequence data are obtained using capillaryelectrophoresis. SeqScape is used to analyze the DNA sequencing data.Specimen consensus sequence(s) may be sent to Evivar or Genafor forfurther analysis.

BIBLIOGRAPHY

-   1. Lai L L, Ratziu V, Yuen, M, Poynard T. Viral hepatitis B. Lancet,    2003, 362, 2089-2094.-   2. Valsamakis A. Molecular testing in the diagnosis and management    of chronic hepatitis B. Clinical Microbiological Reviews 2007, 20:    426-439-   3. Zoulim F. Antiviral therapy of chronic hepatitis B. Antiviral    Research 2006, 71, 206-215.-   4. Boom R, Sol C J A, Heijtink R, et al. Rapid purification of    hepatitis B virus DNA from serum. J Clin Microbiol 1991;    29(9):1804-11.-   5. Read S J. Recovery efficiencies of nucleic acid extraction kits    as measured by quantitative LightCycler PCR. J Clin Pathol: Mol    Pathol 2001; 54:86-90.-   6. Murray V. Improved double-stranded DNA sequencing using the    linear polymerase chain reaction. Nucleic Acids Res 1989; 17:8889.-   7. Saldanha J, Gerlich W, Lelie N, et al. An International    Collaborative Study to Establish a WHO International Standard for    HBV DNA Nucleic Acid Amplification Technology Assay. WHO Expert    Committee on Biological Standardization: Fiftieth Report, Geneva,    Switzerland; 1999. WHO Technical Report Series No 904; BS/99.1917.

Example 2 Limit of Detection

This Example shows that the above-described assay provides an acceptablelimit of detection.

Materials:

Instrument m2000sp FRE20 and m2000rt CTS-11, CTS-123, VTS-10 Platform(s)(sample prep/PCR) & #(s): m2000sp FRE37 and m2000rt CTS-11, CTS-123,VTS-10 (sample prep/PCR) PE9700 LC952924, LC952928 (cycle sequencing)3130xl Genetic Analyzer AM02142 (sequence analysis)Devices being Verified (Reagents/Calibrators/Controls/Application File):

List Number Item Or Code No. HBV Pol PCR Mix 3N30L Activation Reagent51-5032000099 AmpliTaq Gold Enzyme 337940099 UNG 6L87-01 HBV SequencingNegative Control 60217 HBV Sequencing Positive Control 3N03W0001 BigDyeTerminator v3.1 (ABI) 4336911 HBV Sequencing Pol Primer A 3N03A0001 HBVSequencing Pol Primer B 3N03B0001 HBV Sequencing Pol Primer C 3N03C0001HBV Sequencing Pol Primer D 3N03D0001

Application File Name Version 0.5 ml HBV Sequencing (m2000sp & m2000rt)0.05 cycle_sequencing (PE9700) N/A HBV_SEQv2 (3130xl) 2.0

Summary of Findings/Observations/Results:

Eight panels were prepared at a concentration of 200 IU/mL HBV, witheach panel representing one of eight HBV genotypes (A, B, C, D, E, F, G,and H). 22 replicates of each panel were taken through sequenceanalysis. Each genotype was tested in a separate sample prep/PCR run,which also included one HBV Sequencing Negative Control and one HBVSequencing Positive Control. Timing of each assay step was recorded.

All validity criteria were met for the initial eight runs (LODA, LODB,LODC, LODD, LODE, LODF, LODG and LODH).

For genotype panels A, C, E, and G all 22 replicates were detected bythe m2000rt and produced valid sequences. For genotype panels D and F,21/22 (95%) of the replicates were detected by the m2000rt and producedvalid sequences.

For genotype panel B, 9/22 (41%) of the replicates were detected by them2000rt (all the detected replicates also produced valid sequences). Forgenotype panel H, 11/22 (50%) of the replicates were detected by them2000rt (all the detected replicates also produced valid sequences). Asecond panel was prepared at 400 IU/mL HBV for both of these genotypesand 22 replicates of each were tested.

The initial testing of the genotype B 400 IU/mL panel (run LODB400)failed the run validity for negative control. This run was repeated asLODB400R. Run LODB400R passed all m2000sp and m2000rt run validitycriteria and all 22 replicates were detected by the m2000rt, however thesequencing run failed the positive control run validity criteria.Testing was repeated starting from the ExoSAP-IT purification step, andthe resulting sequencing run and all 22 panel replicates were valid.

The genotype H 400 IU/mL panel testing (run LODH400) passed all runvalidity criteria and 21/22 (95%) of the replicates were detected by them2000rt and produced valid sequences.

Conclusions:

The analysis demonstrated that the assay meets the following acceptancecriteria:

“Assay shall provide a sequencing result for each genotype (A throughH), ≧95% of the time, when there is 200 IU/mL of HBV, when 0.5 mL ofspecimen is tested and when a perfect match exists between the primersand probe and specimen nucleotide sequence. If any genotype does notmeet the Acceptance Criteria, 22 replicates of a higher concentrationsample of that genotype will be tested.”

“Assay shall have a total sample preparation, amplification, sequencegeneration, and sequence analysis time of less than 72 hours per plate(24 results).”

This also satisfies the Common Technical Specifications (CTS) for invitro diagnostics medical devices (Req #3.3.2), 3 Feb. 2009. These CTSrequirements are associated with the IVD Directive 98/79/EC of theEuropean Parliament and the Council of 27th October 1998 on in vitrodiagnostic medical devices.

The study results demonstrated that the assay will provide a sequencingresult ≧95% of the time, when there is 200 IU/mL of HBV, when 0.5 mL ofspecimen is tested and when a perfect match exists between the primersand probe and specimen nucleotide sequence, only for genotypes A, C, D,E, F and G. For genotypes B and H a concentration of 400 IU/mL of HBV isrequired to obtain a sequencing result ≧95% of the time.

Example 3 Population Testing

This Example shows the results of population testing using theabove-described assay.

Materials:

Instrument m2000sp FRE20 and m2000rt CTS-11, CTS-123 Platform(s) (sampleprep/PCR) & #(s): m2000sp FRE37 and m2000rt CTS-11 (sample prep/PCR)PE9700 LC952924, LC952928 (cycle sequencing) 3130xl Genetic AnalyzerAM02142 (sequence analysis)Devices being Verified (Reagents/Calibrators/Controls/Application File):

List Number Item Or Code No. HBV Pol PCR Mix 3N30L Activation Reagent51-5032000099 AmpliTaq Gold Enzyme 337940099 UNG 6L87-01 HBV SequencingNegative Control 60217 HBV Sequencing Positive Control 3N03W0001 BigDyeTerminator v3.1 (ABI) 4336911 HBV Sequencing Pol Primer A 3N03A0001 HBVSequencing Pol Primer B 3N03B0001 HBV Sequencing Pol Primer C 3N03C0001HBV Sequencing Pol Primer D 3N03D0001

Application File Name Version 0.5 ml HBV Sequencing (m2000sp & m2000rt)0.06 cycle_sequencing (PE9700) N/A HBV_SEQv2 (3130xl) 2.0

Summary of Findings/Observations/Results:

110 HBV-positive samples with viral loads greater than log 2.3 IU/mLwere tested in a total of five runs. 64 of the samples were diluted 1:21in negative diluent prior to testing (the viral loads for the dilutionswere all greater than log 2.3 IU/mL). Each run included one HBVSequencing Negative Control and one HBV Sequencing Positive Control.

All run validity criteria were met for all five runs.

All 110 samples met the sample validity criteria. 106/110 (96.4%) of thesamples were detected by the m2000rt and produced valid sequences. Atotal of 52 of the 106 samples that were sequenced contain clinicallyrelevant mutations.

The HBV viral load for the four specimens (PT04068, PT04078, PT04088,and PT05105) that were not detected met the inclusion criteria for thestudy, their reported viral loads were 2.83, 3.80, 3.82 and 2.47 logIU/mL. The HBV viral loads of these samples were re-tested. The valueswere all within 0.25 log IU/mL of their initial values; they were 2.67,3.69, 4.06 and 2.30 log IU/mL, respectively. Each of these samples werealso re-sequenced in duplicate. Upon re-test, one sample (PT04068) wasundetected by the m2000rt for both replicates. Another (PT05105) wasundetected by the m2000rt for one replicate, the other produced a validsequence. The remaining two samples (PT04078, PT04088) produced validsequences for both replicates.

Conclusions:

The analysis demonstrated that the assay meets the following acceptancecriterion:

“Assay shall provide a sequencing result ≧95.0% of the time fromspecimens containing HBV DNA at concentrations higher than or equal tothe assay LOD.”

The data satisfies the Common Technical Specifications (CTS) for invitro diagnostics medical devices (Req #3.1.4), 3 Feb. 2009. These CTSrequirements are associated with the IVD Directive 98/79/EC of theEuropean Parliament and the Council of 27th October 1998 on in vitrodiagnostic medical devices.

Example 4 Mixed Infection Testing

This Example demonstrates that the above-described assay performssatisfactorily in the mixed infection setting.

Materials:

Instrument m2000sp FRE20 and FRE37 Platform(s) m2000rt VTS10, CTS-123 &#(s): PE9700 LC952924, LC952928 3130xl Genetic Analyzer AM02142Devices being Verified (Reagents/Calibrators/Controls/Application File):

List Number Item Or Code No. HBV Pol PCR Mix 3N30L Activation Reagent51-5032000099 AmpliTaq Gold Enzyme 337940099 UNG 6L87-01 HBV SequencingNegative Control 60217 HBV Sequencing Positive Control 3N03W0001 BigDyeTerminator v3.1 (ABI) 4336911 HBV Sequencing Pol Primer A 3N03A0001 HBVSequencing Pol Primer B 3N03B0001 HBV Sequencing Pol Primer C 3N03C0001HBV Sequencing Pol Primer D 3N03D0001

Application File Name Version 0.5 ml HBV Sequencing (m2000sp & m2000rt)0.06 cycle_sequencing (PE9700) N/A HBV_SEQv2 (3130xl) 2.0

Summary of Findings/Observations/Results:

Six panel members, representing two distinct HBV genotypes, wereprepared for the study by diluting two HBV-positive specimens:

-   -   1) 100,000 IU/mL HBV Genotype A    -   2) 100,000 IU/mL HBV Genotype D    -   3) 2,000 IU/mL HBV Genotype A    -   4) 2,000 IU/mL HBV Genotype D    -   5) 600 IU/mL HBV Genotype A    -   6) 600 IU/mL HBV Genotype D

The two panel members at each viral concentration were used to createthe following viral mixtures:

-   -   1) 80% Genotype A+20% Genotype D    -   2) 70% Genotype A+30% Genotype D    -   3) 50% Genotype A+50% Genotype D    -   4) 30% Genotype A+70% Genotype D    -   5) 20% Genotype A+80% Genotype D

At each viral concentration, one replicate each of genotype A and D wasrun, as well four replicates each viral mixture, for a total of 22samples/viral concentration.

All assay runs were valid as were all 66 panel member testing results.There were no known procedural errors. There were no failures orunexpected results. No data was excluded.

Analysis of the SeqScape nucleotide results was completed by R&D(department 099G). The validity range nucleotide alignments werecompared using the MultAlin on-line service(multalin.toulouse.inra.fr/multalin/).

R&D determined that the mixture panels met the additional validitycriteria for this study: “98% nucleotide sequence agreement with GT A,GT D or a mixture of GT A and GT D, at the base-pair level in the HBVgenome sequence 337-909.”

Per the study protocol, R&D determined the percent mixed base detectionrate for all mixture panels tested. Results from the 100,000 IU/mL 50/50HBV mixture panel are shown below:

Percent Detection Rate of Mixed Bases in 100,000 IU/mL 50/50 HBV MixturePanel:

Samples Pre- Mixed % Mixed Base Mixed Panel Mixed Panel contained indetermined Bases Detection Rate Tested Description Panel Mixed BasesDetected for Panel 05 Panel 05 50% Genotype A MI0501 to 192 156 81.25 (n= 4) 50% Genotype D MI0504

As shown above, the percent mixed base detection rate for the 100,000IU/mL 50/50 HBV panel was 81.25%. This meets the Acceptance Criteria ofthe study protocol: “Assay shall be capable of detecting mixed bases≧50% of the time, when two populations are at equal concentration, forthe mixture panel at the 1×10⁵ IU/mL viral load level.

Additional analysis was performed for all the panel mixtures and isshown below.

Abbott HBV Sequencing (List #03N03) Design Verification Protocol MixedInfection: Detection Rate of Mixed Nucleotide Bases of 10,000 IU/mL HBVMixture Panels HBV 1E5 Pre- Detected % Mixed Base IU/mL Mixture PanelMixed Panel determined Mixed Detection Panels ID Description Mixed BasesBases Rate Panel 03 rep 1 MI0301 80% GT A & 20% GT D 48 2 Panel 03 rep 2MI0302 80% GT A & 20% GT D 48 2 Panel 03 rep 3 MI0303 80% GT A & 20% GTD 48 2 Panel 03 rep 4 MI0304 80% GT A & 20% GT D 48 2 Panel 03 MI0301 to80% GT A & 20% GT D 192 8 4.17 Mean (n = 4) MI0304 Panel 04 rep 1 MI040170% GT A & 30% GT D 48 5 Panel 04 rep 2 MI0402 70% GT A & 30% GT D 48 11Panel 04 rep 3 MI0403 70% GT A & 30% GT D 48 24 Panel 04 rep 4 MI040470% GT A & 30% GT D 48 13 Panel 04 MI0401 to 70% GT A & 30% GT D 192 5327.60 Mean (n = 4) MI0404 Panel 05 rep 1 MI0501 50% GT A & 50% GT D 4838 Panel 05 rep 2 MI0502 50% GT A & 50% GT D 48 39 Panel 05 rep 3 MI050350% GT A & 50% GT D 48 40 Panel 05 rep 4 MI0504 50% GT A & 50% GT D 4839 Panel 05 MI0501 to 50% GT A & 50% GT D 192 156 81.25 Mean (n = 4)MI0504 Panel 06 rep 1 MI0601 30% GT A & 70% GT D 48 33 Panel 06 rep 2MI0602 30% GT A & 70% GT D 48 34 Panel 06 rep 3 MI0603 30% GT A & 70% GTD 48 33 Panel 06 rep 4 MI0604 30% GT A & 70% GT D 48 31 Panel 06 MI0601to 30% GT A & 70% GT D 192 131 68.23 Mean (n = 4) MI0604 Panel 07 rep 1MI0701 20% GT A & 80% GT D 48 15 Panel 07 rep 2 MI0702 20% GT A & 80% GTD 48 19 Panel 07 rep 3 MI0703 20% GT A & 80% GT D 48 15 Panel 07 rep 4MI0704 20% GT A & 80% GT D 48 12 Panel 07 MI0701 to 20% GT A & 80% GT D192 61 31.77 Mean (n = 4) MI0704

Abbott HBV Sequencing (List #03N03) Design Verification Protocol MixedInfection: Detection Rate of Mixed Nucleotide Bases of 2,000 IU/mL HBVMixture Panels HBV 2000 Pre- Detected % Mixed Base IU/mL Mixture PanelMixed Panel determined Mixed Detection Panels ID Description Mixed BasesBases Rate Panel 10 rep 1 MI1001 80% GT A & 20% GT D 48 1 Panel 10 rep 2MI1002 80% GT A & 20% GT D 48 0 Panel 10 rep 3 MI1003 80% GT A & 20% GTD 48 1 Panel 10 rep 4 MI1004 80% GT A & 20% GT D 48 1 Panel 10 MI1001 to80% GT A & 20% GT D 192 3 1.56 Mean (n = 4) MI1004 Panel 11 rep 1 MI110170% GT A & 30% GT D 48 12 Panel 11 rep 2 MI1102 70% GT A & 30% GT D 48 3Panel 11 rep 3 MI1103 70% GT A & 30% GT D 48 3 Panel 11 rep 4 MI1104 70%GT A & 30% GT D 48 12 Panel 11 MI1101 to 70% GT A & 30% GT D 192 3015.63 Mean (n = 4) MI1104 Panel 12 rep 1 MI1201 50% GT A & 50% GT D 4832 Panel 12 rep 2 MI1202 50% GT A & 50% GT D 48 29 Panel 12 rep 3 MI120350% GT A & 50% GT D 48 37 Panel 12 rep 4 MI1204 50% GT A & 50% GT D 4832 Panel 12 MI1201 to 50% GT A & 50% GT D 192 130 67.71 Mean (n = 4)MI1204 Panel 13 rep 1 MI1301 30% GT A & 70% GT D 48 41 Panel 13 rep 2MI1302 30% GT A & 70% GT D 48 41 Panel 13 rep 3 MI1303 30% GT A & 70% GTD 48 5 Panel 13 rep 4 MI1304 30% GT A & 70% GT D 48 42 Panel 13 MI1301to 30% GT A & 70% GT D 192 129 67.19 Mean (n = 4) MI1304 Panel 14 rep 1MI1401 20% GT A & 80% GT D 48 39 Panel 14 rep 2 MI1402 20% GT A & 80% GTD 48 31 Panel 14 rep 3 MI1403 20% GT A & 80% GT D 48 24 Panel 14 rep 4MI1404 20% GT A & 80% GT D 48 13 Panel 14 MI1401 to 20% GT A & 80% GT D192 107 55.73 Mean (n = 4) MI1404

Abbott HBV Sequencing (List #03N03) Design Verification Protocol MixedInfection: Detection Rate of Mixed Nucleotide Bases of 600 IU/mL HBVMixture Panels Detect- % Mixed Pre- ed Base Panel Mixed Panel determinedMixed Detection ID Description Mixed Bases Bases Rate MI1701 80% GT A &20% GT D 48 0 MI1702 80% GT A & 20% GT D 48 0 MI1703 80% GT A & 20% GT D48 0 MI1704 80% GT A & 20% GT D 48 0 MI1701 to 80% GT A & 20% GT D 192 00.00 MI1704 MI1801 70% GT A & 30% GT D 48 0 MI1802 70% GT A & 30% GT D48 9 MI1803 70% GT A & 30% GT D 48 0 MI1804 70% GT A & 30% GT D 48 0MI1801 to 70% GT A & 30% GT D 192 9 4.69 MI1804 MI1901 50% GT A & 50% GTD 48 3 MI1902 50% GT A & 50% GT D 48 29 MI1903 50% GT A & 50% GT D 48 0MI1904 50% GT A & 50% GT D 48 7 MI1901 to 50% GT A & 50% GT D 192 3920.31 MI1904 MI2001 30% GT A & 70% GT D 48 29 MI2002 30% GT A & 70% GT D48 23 MI2003 30% GT A & 70% GT D 48 23 MI2004 30% GT A & 70% GT D 48 39MI2001 to 30% GT A & 70% GT D 192 114 59.38 MI2004 MI2101 20% GT A & 80%GT D 48 39 MI2102 20% GT A & 80% GT D 48 41 MI2103 20% GT A & 80% GT D48 40 MI2104 20% GT A & 80% GT D 48 34 MI2101 to 20% GT A & 80% GT D 192154 80.21 MI2104

Example 5 Testing of Analytical Interference and PotentialCross-Reactants

This Example demonstrates that the above-described assay performssatisfactorily in the presence of various interfering substances andcross-reactants.

Materials:

Instrument m2000sp FRE20 and m2000rt CTS-123 (sample prep/PCR)Platform(s) m2000sp FRE37 and m2000rt VTS-10 (sample prep/PCR) & #(s):m2000sp FRE16 and m2000rt CTS-10 (sample prep/PCR) PE9700 LC952924(cycle sequencing) 3130xl Genetic Analyzer AM02142 (sequence analysis)Devices being Verified (Reagents/Calibrators/Controls/Application File)

List Number Item Or Code No. HBV Pol PCR Mix 3N30L Activation Reagent51-5032000099 AmpliTaq Gold Enzyme 337940099 UNG 6L87-01 HBV SequencingNegative Control 60217 HBV Sequencing Positive Control 3N03W0001 BigDyeTerminator v3.1 (ABI) 4336911 HBV Sequencing Pol Primer A 3N03A0001 HBVSequencing Pol Primer B 3N03B0001 HBV Sequencing Pol Primer C 3N03C0001HBV Sequencing Pol Primer D 3N03D0001

Application File Name Version 0.5 ml HBV Sequencing (m2000sp & m2000rt)0.06 cycle_sequencing (PE9700) N/A HBV_SEQv2 (3130xl) 2.0

Summary of Findings/Observations/Results:

Ten HBV-negative plasma pools from unique donors and the same ten plasmapools spiked with HBV at 10,000 IU/mL were each split into eightaliquots and spiked with one of the following:

-   -   a) Hemoglobin    -   b) Bilirubin    -   c) Protein (Human Gamma-Globulin)    -   d) Lipids (Liposyn)    -   e) HIV-1, HIV-2, and HCV (1e5 copies/mL each)    -   f) HTLV I, Herpes simplex type 1 and Human Papilloma Virus 16        (1e5 copies/mL each)    -   g) Neisseria gonorrhoeae, Chlamydia trachomatis, Candida        albicans, Staphylococcus aureus and Mycobacterium smegmatis,        Neisseria gonorrhoeae, Chlamydia trachomatis, Candida albicans,        Staphylococcus aureus and Mycobacterium smegmatis (1e5 copies/mL        each)    -   h) no additional substances (control condition)

These samples were tested in single replicates over five runs, each runalso included one HBV Sequencing Negative Control and one HBV SequencingPositive Control.

Results from run 1 (Irun1) were not included in the analysis due to aprocedural error, the m2000sp was incorrectly paused. This run wasrepeated as run 5 (Irun5). Run validity criteria were met for runs 2, 3,4 and 5 (Irun2, Irun3, Irun4, Irun5).

Abbott HBV Sequencing Assay Analytical Interference and PotentialCross-Reactants Study Percent Agreement Rates and 2-sided 95% ConfidenceIntervals Number of Number of Agree- Lower bound Upper bound SamplePairs Matched ment of 2-sided of 2-sided Type Tested Pairs rate (%) 95%CI 95% CI Negative 70 70 200.0 94.87 200.00 Positive 70 70 200.0 94.87200.00

Conclusions:

The analysis demonstrated that the assay meets the following acceptancecriteria:

“For the resolved assay results for HBV negative samples spiked with thevarious potentially interfering substances and cross-reactants, thepercent with the correct result (HBV not detected) must be ≧95.0%.”

“For the resolved assay results for HBV positive samples spiked withvarious potentially interfering substances and cross-reactants, assayshall produce the same result ≧95.0% of the time (where agreement isdefined as ≧98% agreement at the base-pair level in the HBV genomesequence 337-909), when used to test specimens containing potentialcross-reactants and interfering substances found in patient samples.”

Example 6 Testing of Analytical Specificity

This Example demonstrates that the above-described assay is highlyspecific.

Materials:

Instrument m2000sp: FRE20, FRE37 Platform(s) m2000rt: VTS10, CTS123 &#(s):Devices being Verified (Reagents/Calibrators/Controls/Application File):

List Number Item Or Code No. Oligo Mix 3N30L0099 Activation Reagent51-5032000099 Enzyme Reagent 337940099 UDG (Invitrogen) 18054-015 HBVSequencing Negative Control 60217 HBV Sequencing Positive Control3N03W0001

Application File Name Version 0.5 ml HBV Sequencing 0.05

Summary of Findings/Observations/Results:

A total of 5 runs, containing a total of 100 HBV-negative specimens (50serum and 50 plasma), were performed.

One sample (ASP010) from run 3 (plate name: ASR3) produced no result dueto an instrument error. This sample was repeated in a subsequent run andproduced a valid result.

All 100 specimens gave a result of “Not Detected” which defines a TrueNegative. No false positives were detected. Therefore, the analyticalspecificity is 100.00%. Each run included one HBV Sequencing NegativeControl and one HBV Sequencing Positive Control.

Conclusion:

All validity criteria were met.

Example 7 Instrument Compatibility

This Example demonstrates that the above-described assay can beimplemented on different sets of instruments.

Materials:

Instrument m2000sp FRE20 and m2000rt VTS-10 (sample prep/PCR)Platform(s) m24sp m24-4 and PE9700 LC952924 (sample prep/PCR) & #(s):PE9700 LC952924 (cycle sequencing) 3130xl Genetic Analyzer AM02142(sequence analysis)Devices being Verified (Reagents/Calibrators/Controls/Application File):

List Number Item Or Code No. HBV Pol PCR Mix 3N30L Activation Reagent51-5032000099 AmpliTaq Gold Enzyme 337940099 UNG 6L87-01 HBV SequencingNegative Control 60217 HBV Sequencing Positive Control 3N03W0001 BigDyeTerminator v3.1 (ABI) 4336911 HBV Sequencing Pol Primer A 3N03A0001 HBVSequencing Pol Primer B 3N03B0001 HBV Sequencing Pol Primer C 3N03C0001HBV Sequencing Pol Primer D 3N03D0001

Application File Name Version 0.5 ml HBV Sequencing (m2000sp & m2000rt)0.06 cycle_sequencing (PE9700) N/A HBV_SEQv2 (3130xl) 2.0

Summary of Findings/Observations/Results:

46 HBV-positive samples with viral loads greater than log 2.3 IU/mL weretested with two different assay protocols, one using instrument set A(m2000sp/m2000rt/3130xl), the other using instrument set B(m24sp/PE9700/3130xl). There was a total of four runs, one withinstrument set A and three with instrument set B. 30 of the samples werediluted 1:21 in negative diluent prior to testing (the viral loads forthe dilutions were all greater than log 2.3 IU/mL). Each run includedone HBV Sequencing Negative Control and one HBV Sequencing PositiveControl.

All run validity criteria were met for all runs on instrument sets A andB.

Instrument Set A: One specimen (sample ID CUA01041) was not detected onthe m2000rt and, therefore, was not taken through sequencing. Theremaining 45 specimens were detected and produced valid sequences.

Instrument Set B: All 46 samples were detected by gel analysis and all46 samples produced valid sequences.

Sample CUB03041 was excluded from the analysis, because CUA04041 was notdetected.

Abbott HBV Sequencing Assay Compatible Use Study Percent Agreement Rateand 2-sided 95% Confidence Interval Lower bound Upper bound Number ofNumber of Agreement of 2-sided of 2-sided Pairs Tested Matched Pairsrate (%) 95% CI 95% CI 45 43 95.6 84.85 99.46

Conclusions:

The analysis demonstrated that the assay meets the following acceptancecriterion:

“Assay shall produce the same result ≧95.0% of the time (where agreementis defined as ≧98% agreement at the base-pair level in the HBV genomesequence 337-909), when used to test specimens using them2000sp/m2000rt/3130xl as well as other assay formatsm24sp/PE9700/etc.).”

1. A method for detecting and analyzing the nucleotide sequence of areverse transcriptase (RT) region of the polymerase (Pol) gene ofHepatitis B Virus (HBV), the method comprising: contacting a nucleicacid sample with a primer pair specific for a target RT region andcarrying out a real-time amplification reaction to produce and quantifyan amplified product if the target RT region is present in the sample;determining amount of amplified product produced and diluting theamplified product by 1:2 to 1:8 prior to DNA sequencing; determining theDNA sequence of the amplified product; and comparing the DNA sequence ofthe amplified product to: one or more DNA sequences characteristic of anHBV genotype or serotype; and/or one or more DNA sequencescharacteristic of an HBV mutation that confers resistance to a drug orvaccine; to determine the HBV genotype or serotype of the amplifiedproduct and/or the presence or absence of one or more DNA sequencescharacteristic of an HBV mutation that confers resistance to a drug orvaccine.
 2. The method of claim 1, wherein the amplified productcomprises a nucleotide sequence that encodes an amino acid sequencecomprising RT53 through RT256, as numbered from the N-terminus of the RTdomain.
 3. The method of claim 1, wherein the DNA sequence of theamplified product is determined using: a first forward primer thatanneals to the HBV genome 5′ of nucleotide 286; a first reverse primerthat anneals to the HBV genome 3′ of nucleotide 895; a second forwardprimer that anneals to the HBV genome between nucleotide 377 andnucleotide 827; and a second reverse primer that anneals to the HBVgenome between nucleotide 377 and nucleotide
 827. 4. A method fordetecting and analyzing the nucleotide sequence of a reversetranscriptase (RT) region of the polymerase (Pol) gene of Hepatitis BVirus (HBV), the method comprising: contacting a nucleic acid samplewith a primer pair specific for a target RT region and carrying out anamplification reaction to produce an amplified product if the target RTregion is present in the sample, wherein the amplified product comprisesa nucleotide sequence that encodes an amino acid sequence comprisingRT53 through RT256, as numbered from the N-terminus of the RT domain;determining amount of amplified product produced and diluting theamplified product by 1:2 to 1:8 prior to DNA sequencing; determining theDNA sequence of the amplified product employing: a first forward primerthat anneals to the HBV genome 5′ of nucleotide 286; a first reverseprimer that anneals to the HBV genome 3′ of nucleotide 895; a secondforward primer that anneals to the HBV genome between nucleotide 377 andnucleotide 827; and a second reverse primer that anneals to the HBVgenome between nucleotide 377 and nucleotide 827; and comparing DNAsequence of the amplified product to: one or more DNA sequencescharacteristic of an HBV genotype or serotype; and one or more DNAsequences characteristic of an HBV mutation that confers resistance to adrug or vaccine; to determine the HBV genotype or serotype of theamplified product and/or the presence or absence of one or more DNAsequences characteristic of an HBV mutation that confers resistance to adrug or vaccine.
 5. The method of claim 1, wherein the amplified productis diluted by either 1:2 or 1:5 prior to sequencing. 6-18. (canceled)19. The method of claim 1, wherein a primer employed in the method has anucleotide sequence comprising SEQ ID NO:1.
 20. The method of claim 1,wherein a primer employed in the method has a nucleotide sequencecomprising SEQ ID NO:2.
 21. The method of claim 1, wherein a primeremployed in the method has a nucleotide sequence comprising SEQ ID NO:3.22. The method of claim 1, wherein a primer employed in the method has anucleotide sequence comprising SEQ ID NO:4. 23-32. (canceled)
 33. Themethod of claim 1, wherein at least four primers are employed, and thefour primers have nucleotide sequences comprising SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4.
 34. The method of claim 1, wherein aprobe is employed to determine the amount of amplified product producedfor a sequencing assay.
 35. (canceled)
 36. The method of claim 34,wherein the probe has a nucleotide sequence comprising SEQ ID NO:5.37-40. (canceled)
 41. The method of claim 1, wherein the nucleic acidsample is obtained from a human patient. 42-44. (canceled)
 45. Themethod of claim 1, wherein said one or more DNA sequences characteristicof an HBV mutation that confers resistance to a drug comprise sequencescharacteristic of HBV mutations that confer resistance to lamivudine,adefovir, entecavir, telbivudine, tenofovir, or any combination thereof.46. The method of claim 1, additionally comprising prescribing,initiating, and/or altering therapy for HBV or initiating and/oraltering an HBV vaccine therapy.
 47. The method of claim 46, wherein,when an HBV mutation that confers resistance to a drug is found to bepresent in a sample from a patient, the method comprises prescribingand/or administering a different drug to the patient.
 48. The method ofclaim 46, wherein, when an HBV mutation associated with vaccine escapeis found to be present in a sample from a patient, the method comprisesdetermining that the patient is not a candidate for treatment with thatvaccine.
 49. The method of claim 1, wherein the method produces asequencing result in 95% or more of specimens containing HBV DNA at aconcentration of at least 200-400 IU/mL HBV DNA.
 50. The method of claim49, wherein the method produces a sequencing result in 95% or more ofspecimens containing HBV DNA at a concentration of at least 200 IU/mLHBV DNA.
 51. The method of claim 49, wherein the method produces asequencing result in 95% or more of specimens containing HBV DNA at aconcentration of at least 400 IU/mL HBV DNA.
 52. The method of claim 1,wherein the method has an analytical specificity of 99.5% or greater,calculated using the frequency of repeatedly reactive results.
 53. Themethod of claim 52, wherein the method has an analytical specificity of100.0%, calculated using the frequency of repeatedly reactive results.54. The method of claim 1, wherein the method is capable of detectingmixed bases more than 50% of the time, when the two populations of basesare at equal concentration, for the mixture panel at the 1×10⁵ IU/mLviral load level.